| Hepatitis B virus (HBV), a hepadnaviruses, is a partally double-strain DNA virus. Liver injury is likely due to immune response rather than direct viral action. During the course of chronic HBV infection, about 25%-40% of chronic HBV infection patients may die because of Hepatocarcinoma or liver cirrhosis. Recently, study focus on Host genetic characteristics of different outcomes of chronic HBV infection. Numerous studies indicated that patients'outcomes of chronic HBV infection depend on the inflammatory degree of liver injury,and histological liver damage is correlate with host T lymphocyte immune response and virus. Studies showed that T lymphocyte immune function was regulated by Immune regulator.Recently, several reports focused on immune regulators such as PD-1, Bim and IL-7Rα, which control T lymphocyte immune function. PDCD1 gene mapped on chromosomal region 2q37,and it's gene product Programmed cell death protein -1(PD-1) is one of the negative co-stimulate molecules which belong to CD28/B7 family. It is demonstrated that high expression of PD-1 has been identified as a marker of exhausted T cells in chronic viral infection because PD-1/PD-L1 pathway plays a critical role in inhibiting T cell activation and attenuating T cell responses. Bcl-2–interacting mediator( Bim ) gene Bcl2l11 mapped on 2q13, which is one member of the Bcl2 family. Over expression of Bim could trigger T cell apoptosis by inducing cytochrome C's release. CD127 is localized on the p14-p12 region of chromosome 5, while IL-7Rαprotein is a product of CD127. IL-7Rαexpresses in most of peripheral T cells and plays an important role in T cells maturiry, mainteinance and immunity. It has been demonstrated that HBV-specific T cell dysfunction in chronic HBV infection, and aberrant expression of immunoregulatory protein (Bim, IL-7Rα, PD1,) relate with CD8+T cell immune response. It is worthy to further investigate the relationship between immunoregulatory protein and genetic polymorphisms. Genetic polymorphism is one of the mechanisms for regulating expession levels of gene. Characteristic of human genome is polymorphism, including single nucleotide polymorphism and copy number variations. In 2006,a"Copy number variation"map of human genome from 270 individuals from four different populations, found that a total of 1447 CNV regions were identified and covered 12% of the genome. The resutls implied that CNVs are 5-10 times more different than SNP from two randomly selected genomes. While CNVs have been detected previously,recent studies have focus on Geneome-wide association study, in addition, CNVs'effect on the pathogenesis or susceptibility to many diseases including infectious disease have been found. In recent years, there have been more than 2000 CNVs loci and 6000 CNVs detailed in human genome were publicatied on Database of Genomic Variation. Millions of Functional gene, prediction of human diseases and gene regulator elements locus on copy number variations range, and those copy number variations may affect gene's expression, individual's susceptibility to disease, drug's metabolism and Phenotype diversity. In infectious diseases, numerous studies had demonstrated that genes of CNVs may be related to the susceptibility to HIV/HCV infection.In our study, we measure the copy number and the expression levels of several immunoregulatory genes including PDCD1, Bcl2l11 and CD127, in Chronic hepatitis B virus infection patients with different outcomes , so as to analyze the relationsip between gene copy number variation and gene expression level, and the distribution of those immunoregulatory genes'copy numbers in different outcomes of patients of Chronic hepatitis B virus infection.Objective:To measure the distribution of copy numbers of immunoregulatory gene Bim, CD127 and PDCD1 in different outcomes of Chronic hepatitis B infection., and to analyze immunoregulatory gene copy number variations affects gene transcription in PBMCs and gene expression in CD8+T cells.Method and subjects:(1) A total of 111 subjects, including 19 patients with chronic severe hepatitis B(CSH), 33 patients with asymptomatic HBV carriers(ASCs), 29 with HBV related Primary hepatocarcinoma(PHC) and 30 healthy controls(HC), were recruited. (2) Obtain bioinformation about Bim,CD127,PDCD1 genes from Genbank.(3) Coustruct plasimid with single gene copy number of Bim,CD127,PDCD1 genes and gene sequencing.(4) Coustruct plasimid with a single clone cDNA of Bim,CD127,PDCD-1 genes and gene sequencing(5) Using Applied Biosystem TaqMan Copy Number Assays and Copy number TaqMan Refference Assays to measure gene copy number of Bim(109 cases ), CD127(105 cases ), PDCD-1(106 cases).(6) Using Applied Biosystem TaqMan Gene Expression Assays to detect gene transcription of Bim(111 cases ),CD127(111 cases ),PDCD1(111 cases) in peripheral blood mononuclear(7) Detect gene expression of Bim,CD127,PD-1 on CD8+T cell by Multi Flow cytometry(8) Statistical analyse. The Chisquare test was used to analyze the gene copy nubmer distribution, the Kruskal-Wallis test and the Rank Transformation test was used to compare gene transciption in PBMC. One way analysis of variance used to analyze gene expression on CD8+T cell. Spearman's regression was used to study gene copy number,gene transcription and its expression.Results:(I) Copy number of Genes Bcl2l11,CD127,PDCD1(i) Gene copy numbers of Bim: the copy numbers of Bcl2l11 ranged from 1 to 4 among the total subjects. Chisquare test analyze the distribution of Bcl2l11 copy number, the overall difference between each group was statistical significant. We found a significantly difference between the 2 copies of Bcl2l11 in ASCs and CSH(45.46% VS 21.05%),HC(45.46% VS 16.67%).(ii) Gene copy number of CD127: the copy number of CD127 ranged from 1 to 6 among the total subjects. Chisquare test analyze the distribution of CD127 copy number, the overall difference between each group was not significant.(iii) Gene copy number of PDCD1: the copy number of PDCD1 ranged from 0 to 3 among the total subjects. PDCD1 gene copy number deviated from 0 copy to 3 copies among all samples. In HC group, ASC group, CSH group and PHC group, the percentage of cases with a copy number of 1 was 37.0%, 35.5%, 26.3% and 6.9%, respectively, the percentage of cases with 2 copies number was 55.5%, 58.0%, 63.1% and 82.7%, respectively, and the percentage of cases with 3 copies number was 3.7%, 6.4%, 10.5%, 10.3%, respectively. The different distribution of PDCD1 gene copy number was significant in total samples (χ2=9.583,P=0.022). Compared with HC Group, the percentage of cases with 1 copy number was gradually decreased among ASC, CSH and PHC groups while percentage of cases with more than 1 copy number were increased. Besides, a significant difference showed between PHC group and HC Group.(II) mRNA levels of Bcl2l11,CD127,PDCD1 gene in PBMCs(i) Median of mRNA transciption levels of detected Bcl2l11 gene in PBMC in each group is 0.012 (HC), 0.016(ASCs), 0.007865(CSH), 0.007859(PHC), respectively. Kruskal-Wallis tests indicated that the overall difference between each groups was significant(χ2=20.643,P=0.000). the mRNA expression levels in ASCs group was much higher than CSH and PHC group(P<0.0083), but was no differece compared to HC's. (ii) Median of mRNA transciption level of detected CD127 gene in PBMC in each group is 0.194 (HC), 0.164(ASCs), 0.083(CSH), 0.058(PHC), respectively. Kruskal-Wallis tests indicated that the overall difference between each groups was significant(χ2 =27.608,P=0.000<0.01). the mRNA expression levels in ASCs and HC group was significantly higher than CSH and PHC(P<0.0083).(iii) Median of mRNA transciption level of detected PDCD1 gene in PBMC in each group is 0.00254(HC), 0.00272 (ASCs), 0.00255 (CSH), 0.00133 (PHC), respectively. Kruskal-Wallis tests indicated that the overall difference between each groups was significant(χ2=13.911,P=0.003<0.01). the mRNA expression levels in PHC group was significantly lower than the other three group(P=0.001<0.0083). while the difference between ASC and CSH when compared to Control Group not significant.(III) Protein expression of Bim,IL-7Rα,PD-1 on CD8+T cell.(i) Expression of Bim in CD8+T cells (Mean fluorescence intensity, MFI): the value of MFI of Bim expression in each groups is 199.6±33.80(HC), 182.5±46.93(ASC), 384.6±96.00(CSH), 352.8±110.16(PHC), respetively. One way analysis of variance test indicated the overall difference between each groups was significant(F=49.482,P=0.00). Level of expression in CD8+T cell of CSH and PHC is higher than HC and ASCs, there was signficant differences.(ii) Expression of IL-7Rαon CD8+T cell(Mean fluorescence intensity MFI):the value of MFI of IL-7Rαexpression in each groups is 50.6±19.06 (HC), 69.3±24.43 (ASC), 42.8±13.94 (CSH), 352.8±110.16 (PHC), respetively. One way analysis of variance test indicated the overall difference between each groups was significant(F=9.644,P=0.00). Level of expression of IL-7Rαon CD8+T cell of CSH is higher than HC and ASCs, there was signficant differences.(iii) Expression of PD-1 on CD8+T cell(rate ):the rate of PD-1 expression in each groups is 3.72±0.32 (HC), 7.15±0.76 (ASC), 13.40±3.68 (CSH), 16.13±1.68 (PHC), respetively. One way analysis of variance test indicated the overall difference between each groups was significant(F=9.644,P=0.00). Level of expression of PD-1 on CD8+T cell of CSH is higher than HC and ASCs, there was signficant differences.(IV) Correlationship between gene copy number and gene expression.(i) Rate of single copy number of PDCD1 declines from NC, ASC, CSH and PHC groups, and rate of multi copy number of PDCD1 is increase. Most of NC and ASC patients have single copy number of PDCD1, but in CSH and PHC have multi copy numbers. Expression level of PD-1 in CD8+T cells with multi copy numbers of PDCD1 gene is higher than in CD8+T cells with single copy number of PDCD1 gene.(ii) The result shows the negative relationship between transcription of PDCD1 gene and copy number, but that was not significant.(iii) The distribution of Bcl2l11 copy number in NC, ASCs, CSH, and PHC is different, most ASCs patients have 2 copies of Bcl2l11 gene, and rate of 2 copy number in ASCs is higher than other groups. With increase of Bcl2l11 gene copy number from 1 to 3, mRNA transcription of Bcl2l11 gene is increase, but we didn't found the relationship between gene copy number and protein expression of Bim (iv) The distribution of CD127 copy number in NC, ASCs, CSH, and PHC is not significant differentce.Conclusion(I) PDCD1 gene copy numbers were different in Chronic hepatitis B virus infected patients with different outcomes, and copy numbers of PDCD1 in CSH and PHC patients is higher than ASCs and HC group. With the increased of PDCD1 gene copy number, expression of PD-1 protein is increase. These results show that PDCD1 gene copy number variations may regulate protein expression, high expression of PD-1 may effect on CD8+T lymphocyte immue function.(II) Most asymptomatic HBV carriers have 2 copies of Bim gene, but the clinical significance needs further study.(III) This study didn't found the clear correlationship between gene copy number and mRNA transcription, it is necessary to further elucidate mRNA transcription meachanism. |
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