| Background:Hepatitis B virus(HBV)infection,one of the major viral diseases in the word, especially in China,causes a broad spectrum of liver diseases.It has been documented that there are currently about 350 million people with persistent HBV infection worldwide.Persistent HBV infection has been regarded as a multi-factorial disorder relevant to virus,host age,sex,environment,and concurrent infections with the hepatitis C and D virus,and human immunodeficiency virus(HIV).Segregation analysis and twin studies strongly support the role of host genetic background in determining the course of HBV infection.Actually,the susceptibility to infectious diseases is governed by a number of different factors,such as cytokine production, antigen presentation,and receptor recognition.Of note,genetic susceptibility to persistent HBV infection or HBV clearance are likely polygenic,pertaining to genes such as the genes of human leukocyte antigen(HLA)and class cytokine receptor. It has been reported that killer cell immunoglobulin-like receptor(KIR)genes present diversity in the Chinese population.However,it is unknown whether KIR genes participate in the regulation of HBV infectious process.Killer cell immunoglobulin-like receptors(KIR)are a highly diverse family of receptors expressed by natural killer cells and subsets of T lymphocytes.KIR are glycoproteins of Ig superfamily that modulate cells function upon recognition of MHC class I molecules.In humans,The KIR locus,containing a family of polymorphic and highly homologous genes,maps to chromosome 19q13.42 within the leukocyte receptor complex(LRC).Up to date,18 KIR genes were identified,which were KIR2DL1-5,KIR2DS1-5,KIR3DL1-3,KIR3DS1,KIR1D and two pesudogenes KIR3DP1 (including KIRX and KIRXv)and KIR2DP1(also named KIRZ).Besides allelic polymorphism,haplotype diversity is a significant component of human KIR gene diversity as well.It is the characteristic feature same as the HLA-DR that KIR haplotype diversity is variability in the quantity and quality of the genes they contain. Based on gene content,the haplotypes have been divided into two primary sets, termed A and B,the most functionally relevant distinction between haplotypes A and B is the number of stimulatory receptors present.According to the cytoplasmic domain of the length and the existence of ITIM(immunorecept or tyrosine-based inlibitory motify)or not,KIR receptor divide into inhibition(KIR-L)and activation (KIR-S).The inhibitory receptors have a longer cytoplasmic tail containing immunoreceptor-tyrosine-based inhibitory motifs(ITIM).Activating forms have a relatively short cytoplasmic tail and contain a positively charged residue in the transmembrane region that interacts with immune tyrosine-based activation sequence (ITAM).In humans,inhibitory receptors that recognize classical MHC class I molecules.Theoretically,NK cells and T cells activation may be regulated by one of the two following mechanisms:the presence of and signaling,through activating receptors on a large proportion of effector cells(ie,KIR haplotypes containing many activating receptors)or the presence of inhibitory receptor-ligand combinations that send relatively poor inhibitory signals.Upon interaction with HLA class I molecules expressed on the surface of target cells,KIR genes provide activating or suppressing signals to regulate the activation of NK cells and T cells,thereby playing an important role in antiviral and anti-tumor immunity.Previous studies have demonstrated that KIR genes are involved in the pathogenesis of a variety of diseases,including rheumatoid arthritis,vasculitis,psoriatic arthritis, type 1 diabetes mellitus,and hepatitis C virus.In 2002,Martin etal reported in Nature Genetics that the activating KIR allele KIR3DS 1,in combination with HLA-B alleles that encode molecules with isoleucine at position 80(HLA-B Bw4-80Ile),is associated with delayed progression to AIDS in individuals infected with human immunodeficiency virus type 1(HIV-1).Kakoo etal studied 1053 Hepatitis C virus infected patients and found that genes encoding the inhibitory NK cell receptor KIR2DL3 and its human leukocyte antigen C groupl(HLA-C1)ligand directly influence resolution of hepatitis C virus(HCV)infection.The research data strongly suggested that inhibitory NK cell interactions are important in determining antiviral immunity and that diminished inhibitory responses confer protection against HCV. However up to now,the role of KIR polymorphisms in patients with HBV infection has not been investigated.Therefore,the present study was designed to investigate the KIR gene polymorphisms in a large cohort of 150 chronic hepatitis B patients,251 spontaneously recovered cases,and 412 healthy controls by means of sequence specific primer polymerase chain reaction(SSP-PCR),with special attention given to the relationship between KIRs and the HBV infection or HBV clearance.Objective:To explore whether killer cell immunoglobulin-like receptors(KIR)gene polymorphisms are associated with susceptibility to persistent hepatitis B virus(HBV) infection or HBV clearance.Methods:Study subjects:The 813 samples analyzed for this study comprised 150 samples from patients with chronic hepatitis B(CHB),251 spontaneously recovered(SR) controls,and 412 healthy unrelated adult Chinese recruited from Shandong Provincial Hospital and Jinan Infectious Disease Hospital,both in Jinan,between October 2004 and August 2006.CHB patients were required to have a history of hepatitis B viral infection for more than one year and elevated levels of alanine aminotransferase/aspartate aminotransferase or total bilirubin when recruited.Those who were negative for hepatitis B surface antigen(HBsAg)and positive for both hepatitis B surface antibody(HBsAb)and hepatitis B core antibody(HBcAb)were defined as SR controls.All the recruited subjects had no serological evidence of hepatitis C virus,hepatitis D virus,and HIV coinfection;they had no diabetes, malignant tumor,or any autoimmune diseases.All studies were performed after informed consent was obtained from the subjects.Genome DNA extraction:Genomic DNA sample was extracted from 5mL ethylenediaminetetraacetic acid(EDTA)anticoagulated peripheral blood with a standard salting-out procedure and stored at -20℃before use.Genotyping with SSP-PCR method:KIR genotyping was performed by the sequence-specific primer polymerase chain reaction(SSP-PCR)method in all the recruited subjects.KIR locus typing was performed to detect the presence or absence of a total of 15 KIR loci and two pseudogenes.Among them,8 KIR genes(2DLI, 2DL2,2DL3,2DL4,2DL5,3DL1,3DL2,and 3DL3)were responsible for inhibitory functions and 6 KIR genes(2DS1,2DS2,2DS3,2DS4,2DS5,and 3DS1)for conveying activating signals.The SSP-PCR primers used for the detection of KIR loci were based on primer sites that have been previously described.Among the 32 formatted couple primers(Shanghai BoYa biotechnology Co.Ltd.,Shangai,China), 2DS5,KIR1D,3DP1(X)and 3DP1v(Xv)gene use one couple primers and each of the 14 surplus genes uses two couple primers,so as to ensure a detectable rate of positive gene(KIR genes primer in Table 1).The framework genes(2DL4,3DL2,and 3DL3) served as a positive marker of PCR.PCR was conducted by the Gene Amp PCR system 9700-R(Applied Biosystems,Foster City,CA,USA).Briefly,1μL of genomic DNA was amplified in a volume of approximately 20μL system including 6μL primers - 2μL 10×PCR buffer,1.6μL MgCL2(25,000μM),0.4μL dNTP (10,000μM),0.125μL Taq polymerase(5 U/μL),and 8.875μL dH2O.After the initial denaturation for 1 minute at 96℃,the samples were amplified in the following way:5 cycles of 25 seconds at 96℃,45 seconds at 65℃,and 30 seconds at 72℃;21 cycles of 25 seconds at 96℃,45 seconds at 60℃,and 30 seconds at 72℃;5 cycles of 25 seconds at 96℃,1 minute at 55℃,2 minutes at 72℃;and a prolongation of 10 minute at 72℃. Agarose gel electrophoreses and image scanning.:The PCR products,together with approximately 3μL 100 base pairs(bp)DNA ladder as molecular weight marker (MBI,San Francisco,CA,USA),were electrophoresed on 1.5%agarose gels with bromophenol blue,keeping voltage at 160 V for 30 minutes.After electrophoresis,the agarose gel was scanned and imaged by Alphaimager TM 2200 instrument(Alpha Innotech Corporation,San Leandro,CA,USA)and each sample was genotyped.Haplotype Analysis:Each genotype was given the putative haplotype combination according to the model of Hsu et al.In assigning genes to specific haplotypes,the following assumptions were made:(1)all haplotypes contained KIR3DL3,2DL4,and 3DL2;(2)haplotypes contained either 2DL2 or 2DL3,but not both;and(3) haplotypes contained either 3DP1 or 3DP1 variant(3DP1v),but not both.In the assessment of the KIR haplotypes,group B haplotypes were defined by the presence of one or more of the following genes:KIR2DL5,KIR2DS1,KIR2DS2,KIR2DS3, KIR2DS5,and KIR3DS1.Conversely,group A haplotypes were defined by the absence of all these genes.Statitical analysis:The phenotype frequency(pf,%)of each KIR was calculated as the percentage of positive numbers among all specimens.Genotype frequency(gf) was calculated with the formula gf=1-((1-pf))1/2.Haplotype frequency(%)=positive haplotype numbers/2n,n was the total numbers of the researched people in each group. KIR genotype frequency(gf)differences were tested for significance by two-tailed Fisher exact test orχ2 test.Analyses were performed by the SAS 9.0 statistical package(SAS,Cary,NC,USA).Results:1.In this study,all the tested KIR genes were present in both patient groups and control group in different frequencies.Framework genes KIR2DL4,KIR3DL2, KIR3DL3,and KIRZ were present in all individuals.2.The total carriage frequency of KIR2DL5,KIR2DS2,KIR2DS3,and KIR2DS5 was higher in CHB patients than in health control subjects(P=0.001, P=0.001,P=0.011,and P=0.028 respectively). 3.The total carriage frequency of KIR2DS2 and KIR2DS3 was higher(P=0.001, P=0.014 respectively),while the frequency of KIR2DL5,KIR2DS1,and KIR3DS1 was lower(P=0.001,P=0.001,and P=0.001 respectively)in CHB patients than in SR controls.As for KIR2DS5,there was no significant difference between these two groups(P=0.605)4.The total carriage frequency of KIR2DL5,KIR2DS1,KIR2DS5,and KIR3DS1 in SR controls was significantly higher than in healthy controls(P=0,P=0, P=0.001,and P=0 respectively).There was no significant difference in the frequency of KIR2DS2 and KIR2DS3 between the two groups(P=0.526,P=0.919 respectively)5.Individuals who possessed less than two activating KIR genes were more frequently in healthy control subjects than in CHB patients and SR controls.In contrast,individuals with two or more activating KIR genes were more frequently in CHB patients and SR controls than in healthy control subjects(P=0.028,P=0.000 respectively).6.In the study,32 kinds of the genotypes were observed in the three groups.The frequency of genotypes G,M,FZ1 increased in chronic hepatitis B patients compared with health control subjects(P=0.014,P=0.048,and P=0.048 respectively).The frequency of genotype AH was higher in spontaneously recovered controls than both in chronic hepatitis B patients and healthy controls(P=0.004,P=0.030 respectively). The carriage frequency of genotype G and AH was higher(P=0.005,P=0.030 respectively),while the frequency of AF and AJ was lower in SR controls than in Healthy control subjects(P=0.006,P=0.003 respectively).7.Haplotype 2 was found lower in CHB patients than in healthy controls while haplotype 4 and 8 were found higher.Compared with CHB patients,the frequency of haplotype 5 was higher and haplotype 8 was lower in SR controls.The frequencies of haplotypesl and 2 were lower while haplotypes 4 and 5 were higher in SR controls than in healthy controls8.The frequency of A haplotype was lower in CHB patients and SR controls than in healthy controls(P=0.000,P=0.000 respectively),while the frequency of B haplotype was higher(P=0.000,P=0.000 respectively).There is no significant differences with haplotypes A and B between CHB patients and SR controls.Conclusion:SSP-PCR method was used to detect the KIR genes in health control,CHB and SR patients and analyzed KIR genotypes and halotypes,the results indicated that KIR polymorphisms may be associated with susceptibility to HBV infection or HBV clearance.1.It could be suggested that KIR2DS2 and KIR2DS3 were HBV-susceptive genes: The total frequency of KIR2DS2 and KIR2DS3 was higher in CHB group than that both in health control and SR group,and there was no significant difference between health control and SR group.From these finding we concluded that there was an association between the KIR2DS2 and KIR2DS3 and the persisting of HBV infection.2.KIR2DS1,KIR3DS1 and KIR2DL5,on the other hand,may be protective genes that facilitated the clearance of HBV.The total frequency of KIR2DS1, KIR3DS1 and KIR2DL5 was higher in SR group than that both in health control and CHB group.It could be concluded from these finding that KIR2DS1, KIR3DS1 and KIR2DL5 was facilitated to clearn the HBV.3.KIR genotypes and halotypes may be associated with susceptibility to HBV infection or HBV clearance:The total frequency of genotype AH was higher in SR group than that both in health control and CHB group.It could be concluded from these finding that AH genotype was facilitated to clearn the HBV;The frequency of A was lower in CHB patients and SR controls than in healthy controls,while the frequency of B haplotype was higher.There is no significant differences with haplotypes A and B between CHB patients and SR controls.It indicated that halotypes B people easily infected HBV.
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