Genome-wide Association Study Of Hepatitis B Virus Related Acute-on-chronic Liver Failure And Functional Analysis Of The Associated HLA-DR Locus

Acute-on-chronic liver failure（ACLF）is an extreme condition during the natural history of chronic hepatitis B virus（HBV）infection,with highest morbidity in Chinese and Southeast Asians,characterized by development of hyperbilirubinemia and c oagulation abnormality（INR ≥ 1.5）in a short time once onset.And HBV-related ACLF is a critical condition due to its rapid onset,high mortality and lack of specific treatment.However,the underlying mechanisms for its onset are currently unclear.Recently,several researches about ACLF based on alcoholic liver cirrhosis indicate that the outcomes are associated with the amplitude and order of which the ACLF patients had suffered from,include acute hepatitis flare,inflammatory injury,immune disorder,systematic inflammatory response syndrome（SIRS）,compensatory anti-inflammatory response syndrome（CARS）and regeneration.Systemic inflammatory response is the trigger event of patients with alcoholic liver cirrhosis to develop ACLF progression.Because the animal model of chronic HBV infection is unavailable and the etiology and incentive of ACLF is heterogeneous,the immunopathological processes of HBV-related ACLF are still poorly understood.Previous studies on HBV sequence variation suggested that the mutations on basic core promoter（BCP）region,pre-core and core gene were associated with hepatitis flare and fulminant hepatic failure.However,the same mutations on pre-C or C could be found in different diseases,such as chronic hepatitis,liver cirrhosis and hepatocellular carcinoma,even in asymptomatic carrier（As C）.Actually,whether the virus mutation causes the liver failure or not,the host background plays a key role.Common complex diseases were controled both by several minor gene and the environment.The development of HBV-related ACLF include several key points,such as immune recognition,inflammatory activation,inflammatory amplification,hepatocyte necrosis,SIRS,CARS and organ failure,etc.So,the HBV-related ACLF is also a common complex disease,which also was controled by several minor gene and the environment.We know that the genome-wide association study（GWAS）was a powerful tool to discover and identify the genetic factors of common complex disease.So,we performed a three-stage GWAS study among 1300 HBV-related ACLFs and 2087 As Cs to identify the locus associated with ACLF.Main results about this work:1.A total of 3087 samples（1300 HBV-related ACLFs and 2087 AsCs）were included in the three-stage GWAS analysis.All the samples came from one of the five clinic center,Chqongqing,Beijing,Quanzhou,Zunyi,Nanning.Clinical traits analysis showed that the NLC-ACLF（ACLF without liver cirrhosis）patients seem to be younger（39.5 vs.43.9,p = 3.14×10-11）,higher serum ALT（1153 vs.809 IU/L,p = 1.36×10-11）,lower ascites incidence(58.7% vs.88.4%,p = 9.25 × 10^-35),and faster reaching 1.5 for INR and developing encephalopathy（19.8 vs.24.8 days,P = 1.36 × 10-11;27.3 vs.38.1 days,P = 5.16 × 10-6）than that of LC-ACLF patients（ACLF with liver cirrhosis）.2.We compared 35 cytokines among mild/moderate hepatitis B（MM-CHB）,severe hepatitis B（S-CHB）and ACLF（a total of 1013 serums）,and found six ACLF-specific cytokines: IL-1a,TNF-β,IL-5,IL-10,IL-13 and IL-17.3.In the initial GWAS stage,10 SNPs were associated with HBV-related ACLF at p < 1×10-5 level,which were distributed over three loci（1p31,6p21 and 12p11）.But only 6p21,the human leukocyte antigen（HLA）class II gene region,associated with both NLC-ACLF subgroup and LC-ACLF subgroup.The initial findings were replicated in four independent cohort,and identified rs3129859 in HLA-DR region strongly associated with HBV-ACLF（Additive model,combined P = 7.40×10-19,OR=1.83）.4.Stratification analysis showed that rs3129859 was an independent risk factor for HBV-ACLF,rather than hepatitis flare or HBV reactivation.The risky rs3129859*C allele was associated with the clinical progressions of ACLF.For ACLF and S-CHB patients,individuals who carrying rs3129859 risky C alleles had a significantly higher risk of INR reaching 1.5（p = 3.95×10-4）and higher risk of ascites development（p = 3.03×10-4）at 28 days after admission.In NLC-ACLF,individuals who carrying rs3129859 risky C alleles had a significantly higher 28-day mortality rate（p = 0.03）.5.HLA-DRB1*1202 was the top susceptible HLA allele associated with ACLF（p = 3.94×10-6,OR = 2.05）.The frequency of HLA-DRB1*1202 risk allele varies greatly across ethnic populations,with highest rates in Chinese（5.9-21.7%）and Southeast Asians（6.8-35.3%）,which is consistent with the prevalence rate of HBV-related ACLF worldwide.6.The aliphatic residues Glu at position 28 of HLA-DRβ molecular was the top susceptible HLA acid amino associated with ACLF（p = 3.03×10-6,OR = 1.75）,also the acid amino at position 26,30,32,37,38,85 of HLA-DRβ,position 40,47,50,51,53,56,69,76 of HLA-DQα,and position 45 of HLA-DQβ were associated with ACLF（p < 0.001）.We remodeled the three-dimensional structures of HLA-DR and HLA-DQ proteins,and found all the key amino acid located in the antigen recognition domain.7.Allele haplotype rs3129859C-DRB1*1202-DQA1*0601-DQB1*0301 and amino acid haplotype DRβ-LEHHLLA-DQα-GCVLQdel TL-DQβ-E were risk haplotypes for ACLF.For HBV carrier,individuals who carrying the risky haplotype presented a significantly higher risk for ACLF（additive model,p = 1.16×10-4,OR = 1.94;p = 2.43 × 10-6,OR = 2.10,for allele and acid amino haplotype respectively）.8.Survival analysis showed that the risky allele and haplotype were associated with the clinical progressions of ACLF.In NLC-ACLF,individuals who carrying risky rs3129859C-DRB1*1202-DQA1*0601-DQB1*0301 haplotype had a significantly higher 28-day,90-day and 180-day mortality rate（p = 3.49×10-4,p = 0.003,p = 0.003,respectively）.Similar results were observed within the risky DRβ-LEHHLLA-DQα-GCVLQdel TL-DQβ-E haplotype.9.Through e QTL analysis we found that rs3129859 was associated with expression of HLA class II genes,and GRAIL analysis identified HLA-DR and HLA-DQ genes as the most plausible candidates.Literature analysis showed that the genetic variants associated with ACLF risk were partially overlapped with those in chronic HBV infection.10.We identified 12 amino acid sites of HBV proteins under positive selection,which were significantly associated with HLA-DRB1*1202 risk allele and hepatitis B flare.Epitope prediction revealed that the mutation of these sites could cause virus epitope drift.Conclusion:We performed the first GWAS in HBV-related ACLF,and identified a significantly associated locus in HLA-DR region on chromosome 6p21.32 confer risk of HBV-related ACLF.Our findings highlight the importance of HLA class II restricted CD4+ T cell pathway on the immunopathogenesis of HBV-related ACLF,and implicate “one disease hypothesis” for ACLF in chronic HBV carriers regardless of the presence or absence of unrecognized cirrhosis.