Mutation Of Hepatitis B Virus X Gene In Hepatitis B Virus-associated Glomerulonephritis

Object:HBV showed a trend of globalization, there are about350millions HBV carriers around the world, and there are nearly100millions hepatitis B carriers in China. In addition, and there are about100000people by dignosed as HBV infected patients each year, so HBV is a serious infectious disease for people’s health in our country. HBV can lead to liver cirrhosis and chronic hepatitis, and liver cancerrenal damage caused by HBV, and it can also result in kidney damage. According to the uncompleted statistics data, the incidence of renal disease can be as high as20%. Therefore, HBV-GN is one of the major secondary kidney disease in our country.Many clinical studies have confirmed that HBV replication can cause liver disease progression. Such studies as the relationship between HBV replication and HCC, along with the increase of virus replication, the incidence of HCC was significantly increased with statistical difference. For example, The study found that virus levels in serum as compared with the105copies/ml and HBV negative, the risk of HCC was6.6.HBV replication level are independent risk factors of HCC. Therefore, development of HBV replication and liver diseases are closely related. In addition to liver involvement, kidney is anthor important organ of HBV infection. Although previous studies showed that the immune reaction is main mechanism of renal lesions as HBVAg complex mediated, but the relationship between viral replication and kidney disease was not clear. By theoretical analysis, with the increase of the amount of virus replication in serum, circulating immune complexes formed with probability is increased; Secondly, the virus itself can damage kidney tissue, increase the amount of virus can also lead to increased incidence of disease. Therefore, one of the aim of our study is to investigate the relationship between HBV replication and HBV-GN whether a correlation exists between similar lesions of the liver.HBV genome is only about3200bp, including4open ORF, each with different functions. In the previous studies, the X gene has transactivation function, with the organization of the carcinogenesis. In the study of HBV and HCC, X gene and protein function is clear. The HBV gene and liver cells can be integration, X gene can be trans activation, the mutations of X gene may be one mechanism. In many HCC studies showed T1762/A1764double mutant, gene mutation leads to increased viral replication and invasive enhancement. On the other hand, trans activation of X gene, through the cell signaling pathways, can lead to cell apoptosis, fibrosis, cell proliferation and tumor. Therefore, HBV X gene and liver diseases are closely related, Especially there is a direct relationship between hepatocellular carcinogenesis and X gene. But the correlation between HBV-GN and HBV X gene has not been reported. The aim of our study is to explore wether mutations of X gene in HBV-GN patients or not, the serum HBV-GN in patients with DNA extraction, sequencing analysis of X genes, to explore mutations in the X gene and possible pathogenic mechanisms, as well as the effects of mutations on disease, to provide help for the diagnosis and treatment of diseases.Methods:On the one hand, collection of HBV-GN patients with the first onset, according to the selection criteria:HBV-DNA replication; first kidney disease,24hour urinary protein excretion>3.0g; eGFR>60ml/min/1.73m2; liver function (ALT, AST) and the liver imaging (ultrasonography or CT) of patients is normal; the exclusion of other secondary or primary renal disease; renal biopsy pathology showed hepatitis B virus associated antigen (HBVAg) deposition; without usage of antiviral drugs, glucocorticoids and other immunosuppressive drug. Serum HBV DNA level of replication was determinated by using fluorescence quantitative PCR. According to the serum HBV DNA level of replication, All HBV-GN patients was divided into high, middle and low group. The index of24hours urine protein excretion, serum albumin, blood cholesterol and triglyceride, serum creatinine, complement C3and C4, ALT and AST were colleted. The levels of urinary protein served as prognostic criteria after treatment of6,12,18months in HBV-GN patients. At the same time, to compare different correlation of viral replication in patients with membranous nephropathy pathological staging, and urinary protein and virus replication level. According to the immunohistochemistry results in renal pathology, comparison of different replication group and deposition of HBV associated antigen rate. Finally, all patients with antiviral therapy, through18months of follow-up observation, comparison remission rate difference in kidney disease patients, analysis of viral replication level remission rate effect on the lesions in patients with HBV-GN.On the other hand, non antiviral therapy in patients with HBV-GN, patients with HBV-GN by renal biopsy pathology, and there is no chronic lesions, liver cirrhosis and hepatocellular carcinoma. Asymptomatic HBV carriers was collected as control group. Extraction of serum HBV in patients with DNA, Download HBV DNA sequences from the gene bank, in the conservative region of X gene to design primers, the target gene is located between nt1583-nt1793,211bp. PCR product was confirmed by2%agarose gel electrophoresis, stained with ethidium bromide, divided by the optical density was measured DNA concentration≥50ug/ml, bidirectional sequencing. To find out differences in gene sequence comparison in HBV-GN group, find out the hot spot mutations, and compare with the control group, as well as the possible mechanisms in the pathogenesis of HBV-GN.Result:Part Ⅰ:Relationship between Serum DNA replication, clinicopathological characteristics and prognosis of hepatitis B virus-associated glomerulonephritis with severe protienuria by antiviral therapy1Clinical and pathological characteristics in different viral replication of HBV-GN patientsWith the increase of serum HBV DNA level,24hour urinary protein excretion was increased in different degree, and at the same time, plasma albumin were reduced, cholesterol and triglyceride were increased with the increase of viral replication, the difference has statistical significance. Serum complement levels with increased viral replication has a downward trend but the difference was not statistically significant, serum creatinine increased with the replication of the virus also increased, but the difference was not statistically significant. With the increase of serum HBV DNA replication level, renal pathological damage aggravated. By the analysis of78cases of HBV-MN patients, respectively. According to the level of virus replication and pathological stage, with replication increases, pathological stage also increased, the relationship has statistical significance difference. At the same time, the relationship between HBV DNA replication performed by logarithmic transformation and24hour urinary protein excretion in patients through the statistical analysis, which found that with increasing the replication of the virus titer, urinary protein excretion increased, the difference has statistical significance.2The relationship between HBV antigen immunofluorescence and HBV DNA titer in HBV-MN patientsImmunofluorescence sections with HBsAg, HBcAg and HBeAg, to use Imagerpro6.0analysis system, to measurement of glomerular section integral optical density values of the total and glomerular cross-section area, according to the ratio between the calculated average optical density value (IOD/area SUM). The HBsAg, HBcAg and HBeAg with value immunofluorescence average optical density compared with different HBV DNA replication group. With the increase of the deposition of virus titer, antibody antigen complexes increases, the difference has statistical significance.3Comparison of therapeutic effects of3groups of treatment in follow-up18monthsThree groups of HBV-GN patients were receiving antiviral nucleotide drug therapy, more than18months. Liver function damage and virus mutation does not appear in the follow-up of18months, and no deterioration of renal function of the patients.24hours urinary protein excretion was evaluation criteria for the prognosis of the patients. The average excretion of urine protein in each groups were significantly decreased, the difference was significant. Complement C3and C4levels increased compared with before treatment, but there was no significant difference before and after treatment, there was not significant changes in serum creatinine. The total remission rate of patients in18months in each group had difference. The total remission rate differences are statistically significant in low copy group and high copy group. Part II Mutation of hepatitis B virus X gene in hepatitis B virus-associated glomerulonephritis1Mutations in the X gene and the corresponding amino acid changeThere were42cases of the nucleotide sequence of point mutation in HBV-GN group. There were5synonymous mutations without leading to amino acid substitutions, which respectively were nt1585, nt1591,nt1633, nt1675, nt1715; There were7missense mutations and resulted in the amino acid substitution; there were42cases with missense mutations, missense mutations accounted for58.5%in all mutations, and patients with mutations accounted for84%in HBV-GN group. The remaining8patients without mutations. Nucleotide sequence of the missense mutation occured mainly in the following sites:nt1653, nt1726, nt1727, nt1730, nt1753, nt1762, nt1764, nt1726and nt1727, nt1762and nt1764. nt1726/1727and nt1762/1764were regarded as double mutant. According to the nucleotide sequence, amino acid was in70-139amino acid of X protein. Missense mutations in the corresponding X protein were aa94, aa118, aa119, aa127, aa130, aa131.2Gene mutations in control groupThere were5cases of the missense mutation in the control group, accounting for about8%in all control group, which respectively were nt1632and nt1635, aa87and aa88amino acids undergone corresponding replacement.3quantitative DNA in HBV-GN group and control groupHBV DNA replication were statistically analyzed in HBV-GN group and control group after log transformation into normal distribution data. The replication level had no statistical difference between the two groups.4urinary protein quantitative comparison with and without X gene mutations in HBV-GNThere were not statistical difference in urinary protein quantitative comparison with and without X gene mutations in HBV-GN group.Conclusions:1With the increase of HBV DNA virus replication, urinary protein excretion increased, pathological damage aggravated. There were correlation between the HBV DNA replication and HBV-GN lesions.2With the increase of HBV replication, remission rate decreased with antiviral treatment in HBV-GN patients, clinical remission rate decreased. There were correlation between the HBV DNA replication and clinical remission.3The majority of HBV-GN patients have mutations in some key sites of X gene, which lead to the substitutions of amino acid, urinary protein excretion increase in patients with mutations, suggesting that mutations in these critical sites may play an important role in the pathogenesis of HBV-GN.