A Study Of Methyl-CpG Binding Domain Family In Chronic HBV Infection

PART I Alteration of Methyl-CpG Binding Domain Family in Patients with Chronic Hepatitis BBackgroundHepatitis B virus(HBV)infection is a prevalent and serious public health issue all over the world.There are approximate 240 million people chronically infected worldwide which accounts for more than 650,000 deaths annually.Chronic HBV infection is a dynamic process orchestrated by the complex interplay between HBV replication and host immune response.It has been proved that epigenetic modification,especially DNA methylation,involved in the process of chronic HBV infection and contributed to the outcome of chronic HBV infection.As the "readout" of DNA methylation,methyl-CpG binding domain(MBD)proteins could recognize the methylation of cytosine residues at CpG islands.Moreover,MBD proteins also play the role of transcriptional repressor in coordinating crosstalk between DNA methylation,histone modifications,and chromatin remodel to achieve coherent transcriptional programs.Thus,MBD proteins specially recognize methylated DNA and recruit co-repressors to silence gene expression.Emerging epigenetic studies showed that MBD family played an important role in tumorigenesis.Recent studies also revealed the alteration of MBD family in systemic lupus erythematosus(SLE)and rheumatoid arthritis(RA).However,the epigenetic involvement of MBD family in chronic HBV infection has not yet been elucidated.Whether MBD family could play the role of epigenetic modification in the progression of chronic HBV infection remains unknown.ObjectiveThis present study was aimed to determine the expression of MBD family members in various phases of patients with CHB and evaluate the possible role and associations in the progression of CHB.Methods369 patients with CHB and 14 healthy controls(HCs)were included in this retrospective study.369 CHB patients were enrolled from June 2014 to August 2016 in Department of Hepatology,Qilu Hospital of Shandong University.All patients with CHB were divided into 2 cohorts:223 patients from June 2014 to October 2015 were set as the training cohort;146 patients from November 2015 to August 2016 were set as the validation cohort.In the training cohort,there were 20 patients in immune-tolerant(IT)phase,69 patients in immune clearance(IC)phase,103 patients in inactive or non/low-replicative(LR)phase,and 31 patients in hepatitis B e antigen(HBeAg)negative hepatitis(ENH)phase.Meanwhile,there were 16 patients in IT phase,44 patients in IC phase,67 patients in LR phase,and 19 patients in ENH phase among the patients from the validation cohort.Among all the patients,40 patients underwent liver biopsy in order to assess the liver fibrosis stage and inflammatory grade.34 patients underwent liver biopsy were in the training cohort,while 6 patients were in the validation cohort.Aspartate aminotransferase(AST)-to-platelet(PLT)ratio index(APRI)score was used for evaluating the fibrosis severity of CHB.The mRNA levels of MBD family members from peripheral blood mononuclear cells(PBMCs)in patients with CHB and HCs were determined by quantitative real-time reverse-transcription polymerase chain reaction(RT-PCR).Results1.The mRNA levels of MeCP2,MBD1,MBD2 and MBD4 were up-regulated in patients with CHB compared to HCs.However,no significant difference was found in MBD3 mRNA between patients with CHB and HCs.2.The relationships between MBD family and the severity of CHB:MBD2 mRNA expression was positively correlated with serum ALT level(r=0.151,P=0.028).Moreover,MBD4 mRNA level was positively correlated with serum alanine aminotransferase(ALT)(r=0.18,P=0.008)and AST levels(r=0.208,P=0.003).However,there were no statistical correlations among other MBD mRNA and liver functional parameters in patients with CHB.3.In the training cohort,MBD2 and MBD4 mRNA levels were increased in patients with positive HBeAg.All of the patients with positive HBeAg were subjects with positive HBV DNA.Patients with negative HBeAg were divided into 2 groups according to HBV DNA.They were patients with positive HBV DNA(group a)and negative HBV DNA(group b).We found that the mRNA levels of MBD2,MBD3 and MBD4 were lower in group a than those in group b,separately.4.Alteration of MBD family mRNA in 4 immune phases of CHB:MeCP2,MBD1 and MBD2 mRNA levels were increased in 4 immune phases compared to HCs.Moreover,we observed the highest expression of MBD1 mRNA in IT phase compared to other phases.The highest mRNA expression of MBD2 and MBD4 were shown in IC phase compared to other phases.MBD3 mRNA level was increased in IC phase compared to HCs.While,MBD4 mRNA level was up-regulated in IT,IC and LR phases,except ENH phase,compared with HCs.In addition,the MBD4 mRNA level was higher in LR phase than that in ENH phase.Unfortunately,there were no statistical differences from MeCP2 and MBD3 mRNA in different immune phases.5.The predicative value of MBD 1 mRNA for IT phase in patients with CHB:Binary logistic regression analysis showed that MBD1 mRNA was associated with IT phase.The receiver operating characteristics(ROC)curve showed that the area under the curve of ROC(AUC)of MBD1 mRNA for the predication of IT phase from CHB was 0.732(P<0.001),and the optimal cut-off value was 0.0305 with sensitivity of 75%and specificity of 65.52%.In the validation cohort,the AUC of MBD1 mRNA for the predication of IT phase from CHB was 0.704(P=0.001).The cut-off value was 0.0305 with sensitivity of 81.25%and specificity of 61.54%.6.The predicative values of MBD2 and MBD4 mRNA for IC phase in patients with CHB:MBD2 and MBD4 mRNA were significantly associated with IC phase.In the training cohort,the AUC of MBD2 and MBD4 mRNA for the predication of IC phase from CHB were 0.671 and 0.716,separately.The optimal cut-off values of MBD2 mRNA and MBD4 mRNA were 0.0069 and 0.00099 with sensitivity of 85.51%,63.77%and specificity of 44.81%,75.97%,respectively.There was no significant difference between the AUC of MBD2 and MBD4 mRNA(P=0.3872).To assess the combined influence of MBD2 and MBD4 mRNA levels on the predication of IC phase from CHB,a new variable predicated probability was established according to an equation obtained by binary logistic regression:Variable(MBD2 plus MBD4)= 50.591*MBD2+555.483*MBD4-2.314.ROC analysis showed that MBD2 plus MBD4 has an AUC of 0.736(P<0.001).The optimal cut-off value of variable(MBD2 plus MBD4)was-0.841 with sensitivity of 68.12%and specificity of 69.48%.Then,we analyzed the predicative values of the 3 parameters.We found that the AUC of variable(MBD2 plus MBD4)was significantly higher than that of MBD2 mRNA(P=0.0225).In the validation cohort,the AUC of MBD2 and MBD4 mRNA for the predication of IC phase from CHB were 0.665(P<0.001)and 0.741(P<0.001).The optimal cut-off values of MBD2 mRNA and MBD4 mRNA were 0.0069 and 0.00099 with sensitivity of 86.36%,70.45%and specificity of 42.16%,71.57%,respectively.Furthermore,the AUC of variable(MBD2 plus MBD4)was 0.754 which was higher than that of MBD2 mRNA(P=0.004)in the validation cohort.Therefore,the variable(MBD2 plus MBD4)>-0.841 showed a better predication for IC phase from CHB with sensitivity of 70.45%and specificity of 70.59%than MBD2 mRNA alone.7.Alteration of MBD family mRNA in different liver fibrosis stages and liver inflammation grades:In the training cohort,only MeCP2 mRNA was highest expressed in S3+S4 than other 3 groups.While there were no statistical differences of other members among different fibrosis stages.Furthermore,MBD2 mRNA level was lowest in G2 than that in G1 and G3+G4.However,there were no statistically significant differences of other members among different inflammation grades.8.The predicative value of MeCP2 mRNA for the severity of fibrosis in patients with CHB:In the training cohort,MeCP2 mRNA level was significantly associated with the severity of fibrosis.S<2 was considered as the mild fibrosis,and S?2 was the severity fibrosis.ROC curve showed that the AUC for MeCP2 mRNA and APRI score were 0.761 and 0.716(P=0.707),respectively.Interestingly,when S3+S4 was considered as the severity group,the predicative value of MeCP2 mRNA was better than that of APRI scores(AUC 0.958 VS 0.717,P=0.0342).The optimal cut-off values of MeCP2 mRNA for S3+S4 was 0.0068 with the sensitivity of 100%and specificity of 85.19%.Furthermore,the predication of MeCP2 mRNA for S4 was also superior to APRI scores(AUC 0.946 VS 0.677,P=0.0424).The optimal cut-off values of MeCP2 mRNA for S4 was 0.0084 with the sensitivity of 100%and specificity of 90.32%.Thus,MeCP2 mRNA showed better predicative value for S3+S4 and S4.9.Correlations between the mRNA levels of MBD family in patients with CHB:Spearman correlation showed positive correlations between MeCP2 and MBD2(r=0.243,P<0.001),MBD1 and MBD3(r=0.132,P=0.049),MBD2 and MBD3(r=0.209,P=0.002)in patients with CHB.All these results indicated that there were intricate links between MBD family members in patients with CHB.Conclusion1.Abnormal MBD family member mRNA expressed in patients with CHB.2.MBD 1 mRNA from PBMCs could predict IT phase from CHB.3.MBD2 and MBD4 mRNA from PBMCs could predict IC phase from CHB.Moreover,the combined variable(MBD2 plus MBD4)showed a better predication for IC phase from CHB than MBD2 mRNA alone.4.MeCP2 mRNA from PBMCs showed better predicative value for fibrosis severity of patients with CHB.5.MBD family involves in the pathogenesis of CHB and correlates with disease progression,suggesting the value in evaluating disease severity.PART II Alteration of Methyl-CpG Binding Domain Family in Patients with Acute-on-Chronic Hepatitis B Liver FailureBackgroundChronic hepatitis B virus(HBV)infection includes chronic hepatitis B(CHB),occult CHB,hepatitis B liver cirrhosis,acute-on-chronic hepatitis B liver failure(ACHBLF),and so on.ACHBLF is one of the most serious diseases among all types of chronic HBV infection.ACHBLF is a lethal clinical syndrome characterized by acute deterioration of liver function superimposed on chronic liver disease.It accounts for approximate 80%acute-on-chronic liver failure(ACLF)in China.ACHBLF has rapid progression,poor prognosis and high short-term mortality.Even to this day,it is a critical challenge of medical treatments for ACHBLF.Patients with severe liver diseases may develop into liver failure(LF)during the progression of diseases.They are the patients with LF tendency.Some patients with ACHBLF have rapid progression whose initial presentations are not obvious.Active monitoring and early intervention for these ACHBLF patients may reduce morbidity and mortality.Therefore,it is of particular importance for early and accurate prediction of ACHBLF.Emerging evidences showed that epigenetic modification involved in the pathogenesis of ACHBLF.Previous studies revealed the roles of MBD family in tumorigenesis and autoimmune diseases.In our previous study,we observed aberrant expression of MBD family members in patients with CHB indicating severity of CHB.ACHBLF is quite different from CHB which was characterized with the transition from chronic infection into fulminant situation.So,it is still of great importance to investigate the role of MBD family in ACHBLF.ObjectiveThis present study was aimed to determine the alteration of MBD family in patients with ACHBLF,analyze the possible role of them in disease progression,and evaluate the possibly predicative value for prognosis of ACHBLF.MethodsThe retrospective study included 142 patients with ACHBLF and 80 patients with CHB enrolled from Department of Hepatology,Qilu Hospital of Shandong University.92 patients with ACHBLE enrolled from April 2012 to February 2015 were set as the training cohort.50 patients with ACHBLF enrolled from March 2015 to May 2016 were set as the validation cohort.Model for end-stage liver disease(MELD)score was employed to estimate the severity of ACHBLF.30 healthy volunteers were recruited from Clinical Medical College of Shandong University and set as healthy controls(HCs).The mRNA levels of MBD family members from peripheral blood mononuclear cells(PBMCs)in all subjects were determined by quantitative real-time reverse-transcription polymerase chain reaction(RT-PCR).Results1.The serum levels of albumin(ALB)and prothrombin activity(PTA)were lowest in patients with ACHBLF than those in patients with CHB and HCs.On the contrary,the alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBIL),international normalized ratio(INR)levels in serum were highest in patients with ACHBLF than those in patients with CHB and HCs.The nonsurvivors showed lower ALB and PTA than survivors in both training and validation cohorts.However,TBIL,INR and MELD score were higher in nonsurvivors than those in survivors in 2 cohorts.The rates of hepatic encephalopathy(HE),ascites and spontaneous bacterial peritonitis(SBP)were higher in nonsurvivors than those in survivors.2.The mRNA levels of MBD family in ACHBLF:We compared the mRNA levels of MBD family in the traning cohort,CHB and HCs using Kruskal-Wallis test.In the training cohort,MBD1 and MBD2 mRNA were up-regulated compared with CHB and HCs.In ACHBLF,MBD4 mRNA was down-regulated compared with CHB,but up-regulated compared with HCs.Moreover,MeCP2 mRNA level was higher in ACHBLF than that in HCs.Compared to HCs,MeCP2,MBD1,MBD2 and MBD4 mRNA levels were increased in CHB,Furthermore,we observed the high-expressed MBD1,and MBD2 mRNA,and low-expressed MBD4 mRNA in nonsurvivors compared to survivors.However,there were no significant differences between survivors and nonsurvivors in MeCP2 and MBD3 mRNA levels.3.Correlations between MBD family and clinicopathological features in ACHBLF:In the traning cohort,MBD4 mRNA was positively associated with PTA and negatively associated with TBIL,INR and MELD score.We also analyzed the correlations among MBD family members by Spearman's correlation.We observed the positive correlations between MeCP2 and MBD2 mRNA(r=0.292,P=0.005),MBD1 and MBD3 mRNA(r=0.481,P<0.001)in ACHBLF.4.MBD4 mRNA from PBMCs was independent risk factor for the prognosis of ACHBLF:Cox proportional hazards regression model showed that MELD score,HE and MBD4 mRNA were independent risk factors for the prognosis of ACHBLF.5.MBD4 mRNA from PBMCs could predict mortality of ACHBLF in the traning cohort:The area under the curve of receiver operating characteristics(AUC)of MBD4 mRNA in predicating 1-month mortality was 0.907 which was higher than that of MELD score(0.714,P=0.0291).The result suggested that MBD4 mRNA might be a better predicating variable for 1-month mortality of ACHBLF than MELD score.The optimal cut-off values for MBD4 mRNA and MELD score were 5.365E-4,21.87 with sensitivities of 83.33%,72.22%and specificities of 92.86%,76.69%,respectively.Then,we segregated patients by optimal cut-off values and compared the survival rates.Log-rank analysis showed that patients with MBD4 mRNA<5.365E-4 and MELD score>21.87 had lower survival rates and time than opposite patients.The AUC of MBD4 mRNA and MELD score in predicating 2-month mortality of ACHBLF were 0.816 and 0.811(P=0.9335).The optimal cut-off values for MBD4 mRNA and MELD score were 5.469E-4,21.87 with sensitivities of 68.09%,68.09%and specificities of 93.33%,84.44%,respectively.Log-rank analysis showed that patients with MBD4 mRNA<5.469E-4 and MELD score?21.87 had lower survival rates and time than patients with MBD4 mRNA>5.469E-4 and MELD score<21.87.Finally,we compared the AUC of MBD4 mRNA and MELD score in predicating 3-month mortality of ACHBLF.0.762 and 0.847.were the AUC of MBD4 mRNA and MELD score.The optimal cut-off values for them were 5.469E-4,21.55 with sensitivities of 62.75%,72.55%and specificities of 92.68%,87.8%,separately.Patients with MBD4 mRNA<5.469E-4 and MELD score>21.55 had lower survival rates and time than patients with MBD4 mRNA>5.469E-4 and MELD score<21.55,separately.Unfortunately,there were no statistical differences between predicative values of MBD4 mRNA and MELD score for 2-and 3-month mortalities.6.Validation for MBD4 mRNA from PBMCs in predicating mortality of ACHBLF:In the validation cohort,AUC of MBD4 mRNA were significant higher than that of MELD score in predicating 1-month mortality of ACHBLF(0.939 VS 0.762,P=0.035).Patients with MBD4 mRNA<5.365E-4 and MELD score>21.87 had lower survival rates and time than opposite patients,respectively.In predication for 2-and 3-month mortalities of ACHBLF,there were no significant differences between MBD4 mRNA and MELD score(0.843 VS 0.834,P=0.998)(0.787 VS 0.836,P=0.5037).The lower survival rates and time were observed in patients with MBD4 mRNA<5.469E-4,MELD score?21.87 than opposite patients.Furthermore,patients with MBD4 mRNA?5.469E-4?MELD score?21.55 had lower survival rates and time than patients with MBD4 mRNA>5.469E-4,MELD score<21.55,separately.Conclusion1.Abnormal MBD family member mRNA expressed in patients with ACHBLF.2.MBD4 mRNA expression from PBMCs could predicate 1-,2-and 3-month mortalities of ACHBLF.What' s important was that the predicative value of MBD4 mRNA for 1-month mortality was better than that of MELD score.3.MBD4 mRNA expression from PBMCs might be a promising biomarker for predicting the disease severity and short-term prognosis in ACHBLF.