Med J Chin PLA, Vol. 41, No. 11, November 1, 2016 919 HBsAg HBs HBV S N- [ ] HBsAg HBs( ) HBVS (MHR) N- (HCC)2009 7 2015 6 302 284 HBVPCR HBV S MHR HCC 18 HCCHCC SN- >40 HBsAg > HBeAgMRH N- HBV HCC (OR=4.281 95%CI 1.843~9.941 P=0.001 OR=3.146 95%CI 1.633~6.060 P=0.001 OR=2.097 95%CI 1.010~4.357 P=0.047 OR=4.381 95%CI 1.842~10.417 P=0.001) (ALT) HBs> HBeHBV DNAHCC 18 HCC8 HCC 1~4 N- HBV S MHR N-HCC HBsAg HBs MHRHBV HCC [] N- [ ] R735.7 [ ] A [ ] 0577-7402(2016)11-0919-06 [DOI] 10.11855/j.issn.0577-7402.2016.11.08 hepatitis B virus in HBsAg and anti-hbs coexistent patients QIAO Yan 1,2, XU Zhi-hui 2, LU Shan-shan 2, LI Xiao-dong 2, HUANG Peng-yu 2, LIU Yan 2, ZHAO Li 2, YANG Yue 2, XU Dongping 2*, LI Jin 3* 1 Clinical Medicine College, Guilin Medical University, Guilin, Guangxi 541004, China 2 Research Center for Clinical and Translational Medicine, 3 Department of Medical Administration, 302 Hospital of PLA, Beijing 100039, China * Corresponding author. XU Dong-ping, E-mail: xudongping302@sina.com; LI Jin, E-mail: lijin302@hotmail.com This work was supported by the National Natural Science Foundation of China (81373136, 81572010) and Capital Health Development Research Special Independent Innovation Project (2016-2-5032) [Abstract] Objective To investigate the association of additional N-glycosylation mutation in major hydrophilic region (MHR) of hepatitis B virus (HBV) S gene with the risk of hepatocellular carcinoma (HCC) in HBsAg and anti-hbs coexistent patients. Methods A total of 284 patients with coexistence of HBsAg and anti-hbs were enrolled in this study, who were admitted in 302 Hospital of PLA from July 2009 to June 2016. HBV DNA was extracted from serum samples and subjected to nested PCR for full-length S-gene sequencing. The association of MHR additional N-glycosylation mutation and clinical parameters with HCC occurrence risk was analyzed. Specifically, the additional N-glycosylation mutation was dynamically analyzed pre- and post-hcc occurrence for 18 patients. Results Multivariate analysis showed that age >40 years, HBsAg >median, HBeAg negativity, and additional N-glycosylation mutation in MHR were associated with HCC occurrence for the HBsAg and anti-hbs coexistent patients (OR=4.281, 95%CI 1.843-9.941, P=0.001; OR=3.146, 95%CI 1.633-6.060, P=0.001; OR=2.097, 95%CI 1.010-4.357, P=0.047; and OR=4.381, 95%CI 1.842-10.417, P=0.001). In contrast, ALT, anti-hbs, anti-hbe, and HBV DNA levels had no significant association with HCC occurrence. Dynamical analysis showed [ ](81373136 81572010) (2016-2-5032) [ ] [ ] 541004 ( ) 100039 302 ( ) ( ) [ ] E-mail xudongping302@sina.com E-mail lijin302@hotmail.com
920 2016 11 14111 that the additional N-glycosylation mutation had already developed 1-4 years prior to HCC occurrence in the 8 of 18 observed patients. Conclusion Additional N-glycosylation mutation in MHR of HBV S gene had close association with HCC occurrence in HBsAg and anti-hbs coexistent patients, suggesting that HBsAg and anti-hbs coexistence and additional N-glycosylation mutation together could serve as a predictive indicator for HCC occurrence in chronic HBV-infected patients. [Key words] hepatitis B, chronic; carcinoma, hepatocellular; mutation; N-glycosylation (hepatocellular carcinoma HCC) 3 [1] [2-3] 60% (hepatitis B virus HBV) HBV HCC HBV DNAC CS/S HCC [4-8] HBsAg HBs HCC [9] HBV S (MHR) HBs [10] HBV S MHR N- HBV [11] N- s146-148 NCT N- N-X-T/S (X P ) HBsAg HBsHCC HCCHBV S MHR N- 1 1.12009 7 2015 6 302 284HBV 72 HCC (HCC ) 212 HCC ( HCC ) 18 HCC 1~4( 2.5) [12-13] HIV 1.2 DNA ( ) PCR ( )PCR (ABI ) ( Roche ) ELISA ( ) Olympus AU5400 Light Cycler 480HBV DNA 1.3 1.3.1HBV DNA S PCR DNA Out HBV DNA PCRHBV S PSup3 5'-TC GCAGAAGATCTCAATCTCG-3'(2416 2436) SB1R 5'-AGGTGAAGCGAAGTGCACAC-3' (1577 1596) PSup4 5'-CATAAGGTGGGAAACTTTAC-3'(2466 2485) SB2R 5'-TTCCGCAGTATGGATCGGC AG-3'(1258 1278) 25 l 2 Mix Buffer(1/10 )12.5 l PSup3(1/10 )0.25 l SB1R 0.25 l HBV DNA 10 l H 2 O2 l94 5min 94 40s 57 1min12s 72 2min24s 30 72 10min 50 l2 Mix Buffer 25 l PSup4 0.5 l SB2R 0.5 l4 l H 2 O50 l94 5min 94 40s 57 1min 72 2min 30 72 10min PCR DNA 1.3.2 S NCBI genotyping tool(http://www.ncbi.nlm.nih.gov/ projects/genotyping/formpagex.cgi)hbv/c S ( AB014381)S 1.4SPSS 18.0 ( ) x±s t logistic(or) OR 95% (CI) P<0.05 2 2.1HBV S 284N- 11.3%(32/284)HCC 15 sq129n 129-131NGT sg130n+t131n 130-132NNS st131n+m133t 131-133NST 112-113 KNA insertion 114-116NAS st116n 116-118NST s114-115 NTSTT insertion 115-117NTS st131n+m133t 131-
Med J Chin PLA, Vol. 41, No. 11, November 1, 2016 921 133NST sg130n 130-132NTS st113n+114-116 STT deletion 113-115NST & st131n+ M133T 131-133NST 9 131-133NST53.3%(8/15) HCC 17 sg130n 130-132NTS st131n+m133t 131-133NST st116n 116-118NST &st131n+ M133T 131-133NST st113n 113-115 NST 114-115 TTN insertion NST 5131-133NST 70.6%(12/17) 2.2 HCCHCC HBV S MHR 2.2.1 HCCHCC HCCHBsAg HCC (P<0.01 P<0.05) HBV DNA (ALT) HCC (P<0.01) N- (20.8%) HCC (8.0% P <0.01)HBs (TBIL) (P>0.05 1) 1 284 Tab.1 The clinical features of the 284 patients with HBsAg and HBsAb double positivity Item HCC (n=72) Non-HCC (n=212) P value Male [n(%)] 51(85.0) 107(83.6) 0.161 Age(year) 52.4±9.4 40.7±14.4 0.001 HBsAg (COI) 4765±2856 3964±2502 0.024 Anti-HBs (U/L) 54.2(25.8-140.0) 43.7(21.3-98.3) 0.052 TBIL( mol/l) 18.6(12.4-19.0) 16.8(11.2-32.3) 0.495 ALT(U/L) 39.0(26-39) 61(29-60) 0.002 HBV DNA(log 10 U/ml) 4.77±1.59 5.44±1.68 0.005 Additional N-glycosylation mutation [n(%)] 15(20.8) 17(8.0) 0.003 TBIL. Total bilirubin; ALT. Alanine aminotransferase 2.2.2 HCCHCC HCCHBV DNA ALT HCC (P<0.05) N- HCC (21.7% vs 9.4% P<0.05) HBsAg HBs TBIL (P>0.05 2) Item 2 HCCHCC Tab.2 The clinical features of patients after just age HCC (n=60) Non-HCC (n=128) P value Male [n(%)] 51(85.0) 107(83.6) 0.806 Age (year) 49.87±9.36 48.85±6.45 0.388 HBsAg (COI) 5207(1248-5208) 4380(2028-6264) 0.361 Anti-HBs (U/L) 51.6(24.6-133.5) 43.2(21.0-94.9) 0.262 TBIL ( mol/l) 19.2(13.4-28.0) 16.8(10.4-31.8) 0.647 ALT (U/L) 36.88±10.72 89.65±6.73 0.000 HBV DNA (log 10 U/L) 4.69±1.55 5.27±1.67 0.037 Additional N-glycosylation mutation [n(%)] 13(21.7) 12(9.4) 0.021 2.2.3 HBV HCC HCC >40 HBsAg> HBeAg N- HCC (P<0.01) ALT>40U/L HBV DNA HBs> HBe ( 3) 2.2.4 Logistic HCC logistic >40 HBsAg> HBeAg N- HCC (P<0.05) >40 HCC 40 4.281 HBsAg> HCC 3.146N- N- 4.381 3 Tab.3 Univariate analyses of risk factors for predicting HCC development Item HCC (n=72) Non-HCC(n=212) OR (95% CI) P value Male [n(%)] 58(80.6) 174(82.0) 0.095(0.458-1.788) 0.773 Age>40 years [n(%)] 64(88.9) 128(60.4) 5.677(2.592-12.436) 0.000 HBsAg>median 49(68.1) 94(44.3) 2.583(1.466-4.553) 0.001 Anti-HBs>median 40(55.6) 103(48.6) 1.585(0.863-2.900) 0.307 ALT >40U/L [n(%)] 36(47.4) 132(62.3) 0.606(0.354-1.039) 0.067 HBV DNA (log 10 U/ml) level [n(%)] 1(2.23-3.99) 23(31.8) 49(23.1) 1.561(0.866-2.815) 0.137 2(4.00-5.99) 22(30.6) 68(32.0) 0.932(0.523-1.662) 0.811 3( 6) 19(26.4) 76(35.8) 0.642(0.354-1.163) 0.142 HBeAg-negative [n(%)] 31(43.1) 48(22.6) 2.583(1.466-4.553) 0.001 Anti-HBe-positive [n(%)] 39(54.2) 87(41.0) 1.698(0.991-2.909) 0.053 Additional N-glycosylation mutation [n(%)] 15(19.7) 17(8.0) 3.019(1.420-6.419) 0.003 HBsAg median: 4532 COI; Anti-HBs median: 48U/L
922 2016 11 14111 HBeAg HBeAg 2.097 ( 4) 2.3 HCC N- 4 logistic Tab.4 Results of logistic regression analysis Item OR 95% CI P value Male 0.948 0. 433-2.076 0.948 Age >40 years 4.281 1.843-9.941 0.001 HBsAg > median 3.146 1.633-6.060 0.001 Anti-HBs > median 1.585 0.863-2.910 0.137 ALT >40U/L 0.676 0.335-1.367 0.276 HBV DNA (log 10 U/ml) level [n(%)] 1(2.23-3.99) 1.642 0.566-4.762 0.361 2(4.00-5.99) 1.260 0.415-3.825 0.683 3( 6) 1.019 0.311-3.342 0.975 HBeAg negative 2.097 1.010-4.357 0.047 Anti-HBe positive 1.019 0.504-2.065 0.957 Additional N-glycosylation mutation [n(%)] 4.381 1.842-10.417 0.001 HBsAg median: 4532 COI; Anti-HBs median: 48U/L Years before diagnosis of HCC 5 4 3 2 1 P1 P2 P3 P4 P5 P6 P7 P8 2.3.1HCC HBV18 HCC ( 2) 8 HCC 1~4 1 N- 2.3.2 S MHR N- 8 4(P2 P3 P5 P6) HCC MHR N- HCC 3(P1 P7 P8) HCCMHR N- HCCN- HBs P4 HCC MHR N- HCCN- HBs P2HBsAg HBsAg HBs HBsAg HBs MHR N- HCC ( 1) Diagnosis of HCC Year after diagnosis of HCC 1 Age/Gender N-glycosylation mutation 82/M 130-132 GTS NTS 48/M 115-116 insertion NGT 70/M 131-133 TSM NST 59/M 129-131 QGT NGT 57/M 115-116 insertion NTS 61/M 131-133 TSM NST 56/M 116-118 TST NST 60/M 116-118 TST NST, 131-133 TSM NST 1N- HBsAg HBs HCC Fig.1 Dynamical observation of additional N-glycosylation mutation in 8 HCC patients with coexistence of HBsAg and anti-hbs. 130-132 GTS NTS, 115-116 insertion NGT, 131-133 TSM NST, 129-131 QGT NGT, 115-116 insertion NTS, 116-118 TST NST;. HBsAg positive,. Anti-HBs positive 3 HBV S(S )S [14] HBsAg 3 N ( 1~98) MHR( 99~169) C( 170~226) MHR ( 124~147) S SMHR N- (N-X-T/S X P) MHR s146~148 N- N- N- [15-16] HBsAg HBs HBV 5% [17] Jang [18] 755HBVHBsAg HBs HCC HBsAg [22.9%(11/48) vs 7.9%(56/707) P=0.002] Seo [9] 1042HBV 5 10 15 HCC (12.7% 23.4% 69.4% vs 4.9% 13% 20.6%) HBV S MHR N- (11.3% vs 2.9% P<0.01) HCC N- 46.9% 22.6% (P<0.01) [19] HBsAg HBs HCC
Med J Chin PLA, Vol. 41, No. 11, November 1, 2016 923 HCC S MHR N- HCC (χ 2 =8.827 P=0.003) (χ 2 =5.353 P=0.021) >40 HBsAg> HBeAgHBV MRHN- HBV HCC (OR=4.281 95%CI 1.843~9.941 P =0.001 OR=3.146 95%CI 1.633~6.060 P=0.001 OR=2.097 95%CI 1.010-4.357 P=0.047 OR=4.381 95%CI 1.842-10.417 P=0.001) ALT HBV DNA HBeHCC HBsAg N- HBsAg HBs DNA HCC [20] Liu [21] HBV N- HCC HBV DNA HBe HCCHCC 8 HCC S MHR N- 8 130-132 GTS NTS 115-116 NGT 131-133 TSM NST 129-131 QGT NGT 115-116 NTS 116-118 TST NST 6N- HCC N- HBsAg HBsAg HBs HBsAg HBs S MHR N- N- HCCN- HBsAg HBs HCC N-HCC HBV S N- HBV S N- HCC HCC HBV S N- HCC HBsAg HBs HBV S MRHN-HCC S N- HCC HCC [1] Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma[ J]. Lancet, 2003, 362(9399): 1907-1917. [2] LinZ,WangHM,FuJL,et al. Study on the correlation between CD4+ cytotoxic T and course of primary hepatocellular carcinoma[ J]. Med J Chin PLA, 2013, 38(2): 137-140. [,,,. CD4+T [J]., 2013, 38(2): 137-140.] [3] Tang SH, Huang QY, Zhou JM, et al. Risk factors of cholelithiasis in patients with hepatitis B virus-associated chronic liver diseases[ J]. Med J Chin PLA, 2014, 39(6): 485-488. [,,,. [J]., 2014, 39(6): 485-488.] [4] Bouchard MJ, Sonia NM. Hepatitis B and C virus hepatocarcinogenesis: lessons learned and future challenges[ J]. Cancer Lett, 2011, 305(2): 123-143. [5] Bruix J, Sherman M, American Association for the Study of Liver Diseases. Management of hepatocellular carcinoma: An update[ J]. Hepatology, 2011, 53(3): 1020-1022. [6] Qu L, Kuai X, Liu T, et al. Pre-S deletion and complex mutations of hepatitis B virus related to young age hepatocellular carcinoma in Qidong, China[ J]. PLoS One, 2013, 8(3): e59583. [7] Quetier I, Brezillon N, Revaud J, et al. C-terminal-truncated hepatitis B virus X protein enhances the development of diethylnitrosamine-induced hepatocellular carcinogenesis[ J]. J Virol, 2015, 96 (Pt 3): 614-625. [8] RenXQ,XuZH,LiuZG,et al. Mutation and significance of HBV precore and basal core promoter in 958 patients with chronic hepatitis B[ J]. Med J Chin PLA, 2009, 34(6): 663-665. [,,,. 958 HBV C/BCP [J]., 2009, 34(6): 663-665.] [9] Seo SI, Choi HS, Choi BY, et al. Coexistence of hepatitis B surface antigen and antibody to hepatitis B surface may increase the risk of hepatocellular carcinoma in chronic hepatitis B virus infection: A retrospective cohort study[ J]. J Med Virol, 2013, 86(1): 124-130. [10] Liu Y, Zhang L, Zhou JY, et al. Clinical and virological characteristics of chronic hepatitis B patients with coexistence of HBsAg and Anti-HBs[ J]. PLoS One, 2016, 11(1): e0146980. [11] Yu DM, Li XH, Mom V, et al. N-glycosylation mutations within hepatitis B virus surface major hydrophilic region contribute mostly to immune escape[ J]. J Hepatol, 2014, 60(3): 515-522. [12] Society of Infectious Diseases and Parasitology and Society of Hepatology of the Chinese Medical Association. Management scheme of diagnostic and therapy criteria of viral hepatitis[ J]. Chin J Hepatol, 2000, 8(6): 324-329. [. [J].
924 2016 11 14111, 2000, 8(6): 324-329.] [13] Chinese Society of Infectious Diseases, Liver Failure and Artificial Liver Group; Severe Liver Disease and Artificial Liver Group. Guideline for diagnosis and treatment of liver failure[ J]. Chin J Hepatol, 2013, 16(3): 210-216. [,. [J]., 2013, 16(3): 210-216.] [14] Chen JH, Liu Y, Xu ZH, et al. Characteristics of S gene mutation in patients with occult HBV infection[ J]. Med J Chin PLA, 2015, 40(3): 178-183. [,,,. S [J]., 2015, 40(3): 178-183.] [15] Renszel KM, Traister R S, Ly nch WP. Unique N-linked glycosylation of CasBrE Env influences its stability, processing, and viral infectivity but not its neurotoxicity[ J]. J Virol, 2013, 87(15): 8372-8387. [16] Zai J, Mei L, Wang C, et al. N-glycosylation of the premembrane protein of Japanese encephalitis virus is critical for folding of the envelope protein and assembly of virus-like particles[ J]. Acta Virol, 2013, 57(1): 27-33. [17] Pondé RA. The underlying mechanisms for the "simultaneous HBsAg and anti-hbs serological profile"[ J]. Eur J Clin Microbiol Infect Dis, 2011, 30(11): 1325-1340. [18] Jang JS, Kim HS, Kim HJ, et al. Association of concurrent hepatitis B surface antigen and antibody to hepatitis B surface antigen with hepatocellular carcinoma in chronic hepatitis B virus infection[ J]. J Med Virol, 2009, 81(9): 1531-1538. [19] Lu SS, Li XD, Luo SD, et al. Implications of newly-added N-glycosylation mutation of hepatitis B virus S-gene in patients with coexistence of HBsAg and antihbs[ J]. Med J Chin PLA, 2016, 41(5): 351-357. [,,,. HBsAg HBs S N- [J]., 2016, 41(5): 351-357.] [20] Teresa P, Irene C, Francesca S, et al. Hepatitis B virus PreS/ S gene variants: pathobiology and clinical implications[ J]. J Hepatol, 2014, 61(2): 408-417. [21] Liu W, Cao Y, Wang T, et al. The N-glycosylation modification of LHBs effects on endoplasmic reticulum stress, cell proliferation and its secretion[ J]. Hepat Mon, 2013, 13(9): e12280. ( 2016-09-082016-10-09) ( )