| Objective Previous studies suggested that HBV nucleotide mutations may be related to exacerbations of hepatitis B hepatitis, but the relationship between the occurrence of ACLF and HBV mutations in patients with chronic hepatitis B is not clear. The existing research suggested the mutations in pre-C region may be involved in occurrence of liver failure, but not conclusive. Hence the need for further research to clarify whether HBV mutations associated with ACLF and to identify new mutations associated with ACLF.Methods The longitudinal and cross-sectional studies were employed. All serum samples were from serum bank of 302 hospital stored at-80℃.Longitudinal comparison:Serum samples from the pre-ACLF stage, as well as during the occurrence of ACLF were obtained from 4 patients (P1-P4); we also collected serum samples from chronic hepatitis stage and liver cirrhosis stage from 2 patients (P5, P6) as control. HBV DNA was extracted and amplified and ligated into modified pCR2.1 plasmid. Of each individual,3-4 cloned HBV DNAs were sequenced and the sequences were aligned between pre-ACLF and post-ACLF and /or pre-Cirrhosis and post Cirrhosis.Cross sectional comparison:HBV DNAs were extracted from serum samples from 166 patients including ACLF(n=46), chronic hepatitis (CHB n=45) 45, LC(n=65), HCC(n=27), and BCP/Pre-C and C region was sequenced and analyzed.Results⑴Longitudinal analysis showed that there were more longitudinal nucleotide mutations in patients with ACLF than patients with LC. Longitudinal nucleotide mutations presented in more than 1 ACLF patients were nt53, nt1846 and nt1896. Longitudinal nucleotide 1913 mutation was also seen in P2, and C1913A mutation was seen in P1 and P2.(2)Cross-sectional study showed that the rate of nt1753, nt1762, nt1764, nt1846, nt1896 and nt1913 mutations were significant different between 4 groups (P<0.01). Between group ACLF and LC, mutation rates of nt1846 and nt1913 had significant difference (P<0.01) and between group ACLF and HCC, nt1762,1846 and nt1913 had significant difference (P<0.05). A1762T/G1764A was the most common combined mutation with positive rate of 70.5% (117/166); other double mutations were A1846T/G1896A [25.9%(43/166)], A1846T/C1913(A or G) [18.7%(31/166)], A1752G/C1799G [10.8% (18/166)], etc. Also we found that the number of mutations had a relation to the progress of the disease, and HBV from ACLF patients presented more mutations. HBV DNA levels were lower in HBV with nt1753, nt1764, nt1846, nt1896 and nt1899 mutations compared to wild type. And the more nucleotide mutations, the lower HBV DNA level. Furthermore, we found that HBeAg negative patients had more G1896A, G1899A, T1753 (G or C) A1846T, C1913 (A or G) and G1915 (A or C) mutations than HBeAg positive patients. And mutations at ntl752, nt1799 and nt1846 were seen more frequently in HBV genotype B, while mutations at nt1753, nt1762, nt1764 and nt1915 were predominant in genotype C. There were no significant differences of BCP/Pre-C mutations between ACLF patients based on CHB with patients based on LC Multivariate analysis showed that nt1913(C→A or G) and nt1846(A→T) are independent factors for the ACLF [Exp(B)s=3.875 and 3.151, respectively; P=0.017 and 0.041, respectively].Conclusion Nucleotide mutations of BCP/Pre-C are closely related to the progress of HBV-related liver diseases. And mutations at nt1846, nt1896 and nt1913 are possibly related to ACLF. A 1846T and C1913 (A or G) mutations are indenpendant factors for ACLF. |
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