Characteristics Of HBV Reverse Transcriptase Gene And S Gene Of Chronic Hepatitis B Patients Experienced With Nucleoside Analugues

BackgrondDespite the wide use of an effective vaccine, over350million people are chronically infected with chronic hepatitis B (CHB). Some of them developed decompensated liver disease, cirrhosis or hepatocellular carcinoma (HCC). In2006, the hepatitis B epidemiological survey shows that the HBsAg-positive rate of the population aged from1to59years was7.18%, while it was0.96%in children under5years old in our country. The nucleos(t)ide analogues (NAs), including lamivudine (LAM), adefovir dipivoxil (ADV), telbivudine (LDT) and entivavir (ETV), have been approved for treatment of chronic hepatitis B (CHB) due to high potency against HBV, but their antiviral efficacy are negated by the development of resistant mutations. As the polymerase gene overlaps with the HBV surface ORFs, the antiviral resistance mutations can lead to corresponding surface gene mutations resulting in the change of HBsAg antigenicity and vice versa.HBsAg is the main envelope protein of HBV virons and target of neutralization antibody either by natural or passive immunization. However, the core of HBsAg is the major hydrophilic region (MHR) which refers to amino acids99-169, especially to "a" determinant region (aa124-147). The mutation of this region can lead to false negative HBsAg, vaccine immunization failure and also increase the rate of occult hepatitis B infection. In vitro and animal research indicated that nucleotide change of sW182*/rtV191I, sW172*/rtA181T and sW196*/rtM204I in the surface gene of hepatitis B virus leading to truncated surface protein is associated with progression of hepatocellular carcinoma (HCC). This can have significant clinical and public health implications. However, the data regarding to genotype and surface gene mutation is rare, especially in patients with resistance.Objective1. To explore the relationship of genotype and resistant mutation patterns in patients with NAs experienced.2. To explore the characteristics of the resistant associated S gene and S gene stop codon mutations in patients with YMDD changes.3. To explore the characteristics of the resistant associated S gene and S gene stop codon mutations in patients with ADV resistances.4. To evaluate the characteristics of MHR changes between patients without resistant mutantions, patients with YMDD mutations and ADV resistant patients.5. To explore the character of sequence whose HBV genotype is unsure.Material and methods1. PatientsHBeAg positive or negative chronic hepatitis B patients, who were suspected with genotype resistance, were recruited from three regional hepatology centers including1161patients from Nanfang Hospital,148patients from Henan Provincial People’s Hospital and61patients from the First People’s Hospital of Foshan from2009to2011. Patients coinfected with HIV, HCV and HDV were excluded. Serum samples were kept at-30℃until analysis. 2. HBV DNA amplification and sequence analysisHBV DNA was extracted from200μl serum samples using the QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions.3. Amplification of HBV RT regionThe HBV RT region was amplified by nested PCR and the products of the second round were sequenced by ABI3730automated DNA sequencer (Completed by Invitrogen, shanghai).4. Sequence analysisFor comparison of the nucleotide sequence and deduced amino acid sequence analysis, the reference sequences from genotype A to G and I were got from Genebank and aligned with our sequences data using CLUSTAL W. Phylogenic analysis was also conducted to determine the HBV genotype using Neighbor-joining algorithm. The genotype, resistant mutation and S gene changes were also analyzed using online software (.http://lifecenter.sgst.cn/hbv/cnv/search, http://hivdb.stanford.edu/HBV/HBVseql and http://hbv.bioinf.mpi-inf.mpg.de/index.php). Full-length sequence was amplified for genotype determinant and analyzed with NCBI genotype tools.5. Statistical AnalysisFor categorical variables, the Chi-Square test or Fisher’s exact was performed. All data was analyzed using SPSS (version13.0; SPSS, Inc, Chicago, IL). Significant difference was determined when P value was less than0.05(two-tailed).Results1. Characteristics of HBV genotype and reverse transcriptase mutational patterns1.1Genotype analysis A total of773patients including342(44.24%) patients with genotype B,428(55.37%) with genotype C,1patients with genotype D and2patients’ genotype not confirmed, were PCR positive and successfully sequenced. Full-length sequence was amplified and analyzed by the NCBI genotyping tools in one patient. We detected that the full-length sequence was recombinant with several other genotype region and with98%similarity with X/C recombinant in Guangxi, China. And recently the X/C recombinant was defined as genotype "I" by others.1.2Mutational patterns in patients with YMDD mutationsAt last,211patients were detected with YMDD mutants and constituted13mutational patterns. We found103(48.82%,55.34in genotype B vs.42.72%in genotype C, P=0.148, Χ2=2.096) patients infected with rtM204I mutation virus and51.18%patients with rtM204V. More rtM204V+rtL180M mutation was distributed in genotype B (34.91%in C vs.12.38%in B, P<0.001, Χ2=14.801). In contrast, more rtM204I+rtL180M was found in genotype C than genotype B (80%vs.20%, P=0.004, X2=8.08). Interestingly, rtM204V+rtL180M+rtV173L mutations were only found in genotype C(P=0.001).1.3Mutational patterns in patients with ADV resistancesAdefovir resistant mutation points, including rt181T/V/S/G/I and rtN236T/V, were discovered in82(10.61%) of773patients, including31in genotype B and51in genotype C. A total of14kinds of adefovir resistant patterns, including rtA181T, rtA181V, rtA181T/V+rtN236T and rtN236T were discovered. There was no significant difference in respect to these mutation patterns in genotype B and C, except for rtN236T mutation which distributed more in genotype B than C (P<0.001, Χ2=12.794). rtA181S/I, rtA181G+rtT184G, rtA181T+rtM250L, rtMA181T+rtN236T+rtM250L, rtA181V+rtL180M and rtA181T+rtN236T+rtV173L were also detected in our patients. 2Characteristics of resistant associated S gene changes2.1Resistant associated S gene change in patients with YMDD mutationsRtM204I mutation could lead to sW196L/S/stop mutations in the hepatitis B surface gene. Among the134patients with rtM204I variants,110patients (genotype B/C,45/65) lead to sW196L change. While17patients in genotype B and5patients in genotype C harboring rtM204I variants resulted in sW196S change (P=0.002, X2=9.192). And only2patients with rtM204I mutations lead to sW196*change. In comparison, rtM204V mutation only leads to sI195M mutation. Interestingly, sE164D/rtV173L mutation was detected only in genotype C patients (P<0.001).2.2Characteristics of S gene changes in patients with ADV resistanceAs previous studies, rtA181T could not only lead to sW172*mutation in the S gene, but also sW172V and sW172L mutations.6patients with sW172L/rtA181T mutation were detected in genotype C. Other mutations, including sL173F/rtA181V, SL173V/HA181G, sW172*/rtA181I and sW172C/rtA181S mutations, was also detected in these patients. sW172*/rtA181I mutation was not reported in previous study.3Characteristics of MHR mutations in all patientsThe characteristic of MHR mutation was presented both in percent of patients with mutation and number of amino acid substitution. Firstly, MHR mutations were detected in60.66%(128/211) of YMDD mutational patients versus75.6%(62/82) in ADV resistant patients versus44.7%(211/472) in patients without resistant mutation. More amino acid (aa) substitutions in amino acids124-137, which considered the number of mutation points per100amino acids in each sequence, were found in genotype B (1.46in patients without NAs resistance vs.2.56in YMDD mutational patients vs.2.07in ADV resistant patients per100amino acids, P=0.037, Χ2=6.576) than in genotype C (2.57in patients without NAs resistance vs.2.52in YMDD mutational patients vs.2.52in ADV resistant patients per100amino acids, P=0.993, X2=0.014). Similarly, there was also statistically significant difference regarding amino acids149-169region (0.5in patients without NAs resistance vs.1.13in YMDD mutation patients vs.0.84in ADV resistant patients per100amino acids, P=0.008, X2=9.546)4Characteristics of S gene stop mutations5gene stop codon mutations including sS53*, sS61*+sC69*, sW74*, sW196*, sY100*+sW196*, sY206*and sL216*, were identified in8patients (4in patients with genotype B and4in genotype C) in patients with YMDD mutations. And only2(1.49%,2/134) patients with rtM204I mutations lead to sW196*change. No significant difference was detected in regarding to S gene stop codon distribution between genotype B and C in patients with YMDD mutations.In ADV resistant patients, most of the rtA181T mutation was corresponding with sW172*change. And6mutational patterns including sW172*, sW172*+sW199*, sW172*+sC76*, sW172*+sC69*, sW172*+sW182*and sY200*+sW201*were identified. While only sW172*mutation, which was detected in25patients, was the most mutations. sW172*+sW199*and sW172*+sW182*was detected in2patients respectively.But8patterns of S gene stop codon mutations including sS53*, sS61*. sC69*, sC69*+sW74*, sW182*, sW196*, sW201*and sW216*were also detected in patients without resistant mutations. sW196*/rtD205N mutation was first reported in our study and it is unclear whether this kind of mutation is associated with resistance.Conclusion1. HBV genotype Ⅰ was detected in our study in2patients and only1was confirmed.2. More rtM204V+rtL180M and rtN236T mutation was distributed in genotype B. In contrast, more rtM204I+rtL180M were mainly found in genotype C. While rtM204V+rtV173L+rtL180M was no exclusively distributed in genotype C. There was no significant difference in respect to rtA181T distribution in genotype B and C.3. Only small number of rtM204I mutation lead to sW196*change and sW196L/S mutations mainly distributed in genotype C.4. RtA181T could not only lead to sW172*mutation in the S gene, but also sW172V and sW172L mutations.5. More MHR mutation was detected in patients with YMDD mutations and ADV resistance than no resistant patients.6. The S gene stop codon mutations were detected both in genotype B and C.