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Genotypic analysis of hepatitis B virus from patients with fulminant hepatitis: comparison with acute self-limited hepatitis
Hepatology Research 26 (2003) 119 /124 www.elsevier.com/locate/ihepcom
Genotypic analysis of hepatitis B virus from patients with fulminant hepatitis: comparison with acute self-limited hepatitis Riho Joh, Kiyoshi Hasegawa *, Miho Ogawa, Kayo Ishikawa, Aiko Iizuka, Takuma Naritomi, Naoko Kanai, Nobuyuki Torii, Etsuko Hashimoto, Naoaki Hayashi Department of Medicine, Institute of Gastroenterology, Tokyo Women’s Medical University, 8-1, Kawadacho, Shinjuku-ku, Tokyo 162-8666, Japan Received 20 August 2002; received in revised form 19 November 2002; accepted 16 January 2003
Abstract Aim/background: There is an increasing evidence that certain hepatitis B virus (HBV) strains may contribute to the pathogenesis of fulminant hepatitis B (FHB). Recently, we reported that genotypes of HBV influence the clinical course of acute self-limited hepatitis B (AHB). In this study, we compared clinical features of FHB between different HBV genotypes and compared the prevalence of each genotype between FHB and AHB patients. Methods: The subjects consisted of seven patients with FHB and 25 patients with AHB. The core promoter and precore region were directly sequenced following polymerase chain reaction, and genotype was determined by restriction fragment length polymorphism analysis of the S gene. Results: Of the seven FHB patients, one had genotype A, one had genotype B, four had genotype C, and one had genotype D. Six of the seven FHB patients were infected by heterosexual contact; one FHB patient who was not infected by heterosexual contact had genotype C. All four FHB patients with genotype C had a short duration clinical course. In one patient with genotype A, the time from onset of hepatitis to hepatic coma was 30 days. These results are similar to those of the patients with AHB, in which clinical course was longer in patients with genotype A than in patients with genotype C. Conclusion: Viral genotype can be used to predict the clinical course of both FHB and AHB. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Fulminant hepatitis; Genotype; Core promoter; Precore
1. Introduction Approximately 1% of patients infected with hepatitis B virus (HBV) develops fulminant hepatitis B (FHB), which is characterized by rapid destruction of the liver parenchyma [1]. In Japan, 34.7% of fulminant hepatitis is associated with HBV infection, including acute onset type and acute exacerbation of chronic hepatitis B (CHB) [2]. The survival rate of HBV-induced fulminant hepatitis is 50 and 30% in patients with acute-onset and chronic HBV infection, respectively. In other words, more than half of the patients with FHB ultimately requires liver transplantation or die of liver failure. The pathogenesis of FHB is not well understood. Previous studies have indicated that complicated interaction
* Corresponding author. Tel.: /81-3-3353-8111; fax: /81-3-52697435. E-mail address: [email protected] (K. Hasegawa).
between viral factors and host immune response may be involved. In previous studies, FHB was found to be associated with the precore stop codon mutation G1896A [3 /11] and two mutations in the core promoter region (A1762T and G1764A) [12,13]. These associations have mainly been found in countries with epidemic hepatitis B. In many reported cases of FHB in non-endemic areas, none of these three mutations was observed [14 /18]. Appearance of precore mutation is influenced by nt 1858, which is the counterpart of nt 1896 in the stemloop structure of pregenome RNA. The base at nt 1858 is cytosine (C) in genotypes A and F, and is thymidine (T) in genotypes B, C, D and E. Mutation from guanine (G) to adenine (A) at nt 1896 occurs readily in genotypes B, C, D and E possibly due to stabilization of the stemloop structure by the matching of adenine to thymidine [19]. In HBV of genotypes B and C, which are the predominant types in Asia, the base at nt 1858 tends to
1386-6346/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1386-6346(03)00011-1
A B C C C C D Heterosexual Heterosexual Heterosexual Unknown Heterosexual Heterosexual Heterosexual Death Death / / Death Death / / / / / / / / 30 5 7 7 7 8 5 /(14)//(41) /(0.9)//(95) /(4)//(84) /(0.9)//(96) /(3)//(51) /(3)//(84) /(1.0)//(91) 1 2 3 4 5 6 7
Clinical data were obtained on admission. a Days from onset to appearance of hepatic coma. b Route of infection.
41.0 8.3 7.1 10.7 4.6 7.9 3.8
527 3.480 4.910 5.008 4.850 8.130 11.060
18.4 12.5 26.0 17.7 16.9 B/10 24.7
/// /// /// /// /// /// ///
HBeAg/anti-HBe (COI) Table 1 Clinical features of seven patients with FHB
The subjects consisted of seven patients with FHB and 25 patients with acute self-limited hepatitis B (AHB). The seven patients with FHB (mean age, 419/ 19 years; four males and three females; Table 1) had been admitted to our hospital and were all diagnosed with acute-onset fulminant hepatitis. The criteria for FHB were development of hepatic encephalopathy, prolongation of the prothrombin time during the course of hepatitis and IgM anti-HBc antibody [21]. All FHB patients had no prior hepatitis. Three FHB patients survived to the end of the follow up period; all three recovered from hepatitis with loss of HBs antigen (HBsAg) and appearance of anti-HBs antibody in the follow up period. In all three surviving FHB patients, chronic hepatitis was excluded by histological examination. Four FHB patients died during the study period: three from liver failure and one (case 1) from multiple organ failure. In case 1, titer of anti-HBc antibody was high, because he was transferred from another hospital 21 days after onset and his anti-HBc antibody titer had not been checked at that hospital. He died after receiving a liver transplant from his father. Histological examination of his explanted liver showed acute hepatitis with submassive hepatic necrosis and no evidence of chronic hepatitis. Hepatitis B e antigen (HBeAg) was positive in three of the four patients who died, and negative in two of the three patients who survived. HBV DNA, which was measured by transcription mediated amplification (TMA), was undetectable in two patients: cases 1 and 2. Cases 1, 2, 6 and 7 were infected from unspecified sexual partners and cases 3 and 5 were infected from their spouses. The 25 AHB patients are described in detail elsewhere [22]. Briefly, we chose 25 patients, whose serum samples were available, from among 27 patients with acute hepatitis B treated at our hospital from 1996 to 2000. The AHB patient group consisted of 23 males and two females, ranging in age from 16 to 48 years (mean age, 329/8 years). AHB was diagnosed according to the following criteria: acute-onset liver dysfunction, IgM
IgM-HBc (LGE/ml)
2.1. Patients
23/M 70/M 28/F 61/M 34/F 49/M 22/F
2. Patients and methods
/3.7 /3.7 8.1 4.2 6.4 7.3 5.3
HBV DNA Daysa Brain edema Outcome Routeb
be T. In genotype A, which is common in European countries, the base at nt 1858 is C [20]. Therefore, it appears that such differences in prevalence of HBV genotype between geographic areas are related to differences in prevalence of the precore G1896A mutation. In this study, we examined viral factors influencing the pathogenesis of FHB by studying the genotype of HBV and the sequences of the core promoter and precore region.
2.7 3.3 3.1 3.1 2.8 3.2 3.2
Genotype
R. Joh et al. / Hepatology Research 26 (2003) 119 /124
Case age/sex T-Bil (mg/dl) ALT (IU/l) PT (%) HBsAg/anti-HBs
120
R. Joh et al. / Hepatology Research 26 (2003) 119 /124
anti-HbcAb-positive, no prior hepatitis, and recovery from hepatitis with loss of HBsAg and appearance of anti-HBs antibody in the follow-up period. 2.2. Determination of genotype The HBV genotype was determined by restriction fragment length polymorphism analysis after polymerase chain reaction (PCR-RFLP), as described by Lindh et al. [20]. Briefly, DNA extracted from patients’ sera was amplified by PCR using the following primers: 5?GTGGTGGACTTCTCTCAATTTTC-3? (sense; nt 256 /278) and 5?-AGGTAAAAAGGGACTCAAGAT3? (anti-sense; nt 796/776). Then, 5 ml of PCR product was mixed with five units of TSP509i, 2 ml of 10 / buffer and 12 ml of water, and then incubated at 65 8C for 3 h. In a separate reaction, 5 ml of PCR product was mixed with five units of HinfI, 2 ml of 10/ buffer and 12 ml of water, and then incubated at 37 8C for 3 h. These mixtures were then resolved on 3% agarose gels and stained with ethidium bromide. The restriction profiles were visually interpreted. 2.3. Amplification of HBV DNA by PCR and sequencing of PCR products Serum (50 ml) was diluted with 50 ml of sterile distilled water and mixed with 100 ml of a mixture containing 25 mM sodium acetate, 2.5 mM EDTA (pH 8.0), 1% SDS, 2 mg/ml of proteinase K and 10 mg/ml of yeast t-RNA as a carrier. Following digestion at 65 8C for 2 h, viral nucleic acids were extracted twice with phenol /chloroform, extracted once with chloroform, ethanol-precipitated and suspended in water. The core promoter and precore regions, consisting of a 1359-bp sequence extending from nt 1435 to 2793, were amplified by PCR using the extracted HBV DNA. The primers were 5?-CGTCGGCGCTGAATCC-3? (sense; nt 1435/1450) and 5?-CGAGGCGAGGGAGTTCTTCTTC-3? (antisense; nt 2793/2771). Then, 5 ml of the extracted DNA was mixed with 45 ml of PCR mixture containing 400 nM of the primers, and amplified by 35 cycles of 94 8C for 1 min, 55 8C for 1 min and 72 8C for 3 min, in an automatic DNA thermal cycler (Perkin/Elmer Cetus, Norwalk, CT). The PCR products were directly sequenced by dideoxy-chain termination using an AutoRead DNA sequencing kit (Pharmacia LKB Biotechnology, Uppsala, Sweden) with the anti-sense primer used in PCR.
3. Results Of the seven FHB patients, one had genotype A, one had genotype B, four had genotype C and one had genotype D. In patients 1 and 2, who had genotypes A
121
and B, respectively, HBV DNA was not detectable by TMA. In contrast, the four patients with genotype C had HBV DNA levels ranging from 4.2 to 8.1 LGE/ml (mean 6.59/1.7 LGE/ml). Patient 1, who had genotype A, had a long-duration clinical course of hepatitis: 30 days from onset to coma. This patient had high total bilirubin and low peak ALT. However, he resisted intensive treatment, including artificial liver support, and eventually died due to complications after liver transplantation. With the exception of case 1, there was no difference in days from onset to coma between patients with genotypes B, C and D. Three patients who died (one patient with genotype B and two with genotype C) exhibited brain edema. Sequence analysis of core promoter and precore region revealed double mutation at nt 1762 and 1764 in all four patients with genotype C, whereas no patients with genotype A, B or D had this double mutation (Fig. 1). This double mutation was detected in four of the 25 AHB patients (Table 2). Only two of the six AHB patients with genotype C had the double mutation at nt 1762 and 1764 (Table 2). No other common mutation in the core promoter region was found in the FHB patients. Thus, mutations in the core promoter region were more prevalent in the FHB patients than in the AHB patients, but this difference was not significant. In the precore region, nt 1858 was thymidine in all four FHB patients with genotype C and the one FHB patient with genotype D. Of these five patients with thymidine at 1858, four had a G-to-A mutation at 1896. Two patients, one with genotype A and one with genotype B, had cytidine at 1858 and guanine at 1896. Only two of the seven AHB patients with thymidine at 1858 had a Gto-A mutation at 1896.
4. Discussion In this study, four of the seven FHB patients had HBV of genotype C and all patients with genotype C had a short course of fulminant hepatitis. Previously, we reported that genotype A was most prevalent in patients with AHB in our hospital, and that the clinical course of acute hepatitis caused by genotype A tended to progress slowly [22]. In this FHB patient with genotype A, the inability to detect HBV DNA by the TMA method on admission may be due to the long interval between onset and admission. Together, these findings suggest the possibility that, in patients with genotype A, weak capacity for recovery from liver injury contributes to the development of subacute fulminant hepatitis. In contrast, acute hepatitis caused by HBV of genotype C was characterized by a shorter course and more severe hepatic injury than acute hepatitis caused by HBV of other genotypes. In this study, the one fulminant hepatitis patient with genotype A had a slowly progres-
R. Joh et al. / Hepatology Research 26 (2003) 119 /124
122
Fig. 1. Upper part: nucleotide sequences of a portion of core promoter region of HBV in seven Japanese patients with fulminant hepatitis, and relation to HBeAg/HBeAb phenotype. Consensus sequences adr and adw are described elsewhere [28]. Letters surrounded by open squares in consensus sequences represent A at 1762 and G at 1764. Lower part: nucleotide sequences of precore region. Letters surrounded by open squares in consensus sequences represent T at 1858 and G at 1896.
sing course, developing hepatic coma 30 days after appearance of the first symptom, which was general malaise. The reason for differences in clinical course of acute and fulminant hepatitis between genotypes is unclear. One explanation is that HBV of genotype A has cytidine at nt 1858, which is the counterpart of nt
1896 in the stem-loop structure of pregenome RNA, and that this inhibits nucleotide substitution from guanine to adenine at nt 1896, in turn inhibiting termination of HBeAg production. In fact, patient 1 had cytosine at nt 1858 and guanine at nt 1896. In patients with CHB, genotype A is significantly more likely to be associated
Table 2 Comparison of sequences of core promoter and precore region between patients with AHB and fulminant hepatitis Genotype (n )
HBeAg
Core promoter (1762/1764)
Precore (1858)
Precore (1896)
/
/
A/G
T/A
ND
T
C
G
A
AHB
A (14) B (5) C (6)
12 4 5
2 1 1
9 4 4
2 0 2
3 1 0
0 4 3
14 1 3
14 5 4
0 0 2
FHB
A (1) B (1) C (4) D (1)
1 0 3 0
0 1 1 1
1 1 0 1
0 0 4 0
0 0 0 0
0 0 4 1
1 1 0 0
1 1 1 0
0 0 3 1
ND: not detectable.
R. Joh et al. / Hepatology Research 26 (2003) 119 /124
with HBeAg-positive phenotype at first examination than other genotypes [23]. Thus, continuous HBeAgpositive phenotype may contribute to long duration of liver injury. The other explanation is that severe acute hepatitis with long clinical course can be controlled by antiviral agents such as lamivudine or interferon, if the therapy is started in the early phase of hepatitis. However, once acute fulminant hepatitis has developed, it is difficult to prevent uncontrolled liver failure. It has been reported that hepatitis of genotype B has a greater tendency to subside over time and is more easily controlled by medication than hepatitis of genotype A or C [24,25]. However, it has been reported that some patients with genotype B have rapidly progressive and severe clinical courses [26]. In these latter cases, part of HBV genotype B was substituted with sequences of genotype C by recombination. This type of genotype B HBV, which is epidemic in southeast Asia and rare in Japan, is called genotype Ba [26]. Based on results of sequence analysis, we classified the HBV of case 2 as genotype BJ (data not shown). It is unclear what relationships exist between subclassification of genotype B and clinical course of acute onset hepatitis B. The present FHB patients had more mutations in both the core promoter region and precore region than AHB patients. The mutations A1762T and G1764A are reportedly associated with fulminant hepatitis in Japan [12]. However, in case 1 (genotype A) and case 2 (genotype B), the nt at 1762 and 1764 were A and G, respectively. Together, genotypes A and B constitute a minority of HBV cases in Japan. Recent studies in the United States and Germany indicate that the A1762T/ G1764A double mutation is not associated with FHB [17,18]. Thus, there appear to be differences between Caucasian and Asian patients in the significance of various sequences in the core promoter region. Recently, it has been reported that double mutations in the core promoter region contribute to enhanced replication of HBV and active liver diseases [27]. In this study, mutations in the core promoter region were only detected in FHB patients with genotype C. However, the number of patients with fulminant hepatitis was too small to determine associations between core promoter sequences, HBV genotype and development of fulminant hepatitis. Therefore, a study with a larger number of FHB patients is needed. In this study, guanine at nt 1896 was converted to adenine in four of the five FHB patients with thymidine at nt 1858. In contrast, G-to-A mutation at nt 1896 was detected in only two of the seven AHB patients with thymidine at nt 1858. These results suggest that G-to-A mutation at nt 1896, which produces a stop codon in the precore region, results from active replication, which is observed in patients with fulminant hepatitis. Cases 2 and 3 (both of which had cytidine at nt 1858) had guanine at nt 1896. Previously, wild type sequence at nt
123
1896 has reportedly been detected in American patients with fulminant hepatitis [14]. Although genotype was not analyzed in that study, we can speculate that differences in HBV genotype distribution between American and Japanese hepatitis patients are responsible for these differences in the frequencies of different sequences at nt 1896 between Japanese and American studies. In conclusion, HBV genotype can be used to predict the clinical course of acute onset hepatitis. However, only a limited number of patients with fulminant hepatitis were examined in this study. A larger study may produce more practical information for use in diagnosis and therapy of acute-onset hepatitis B.
References [1] Sterneck M, Kalinina T, Gu¨nter S, et al. Functional analysis of HBV genomes from patients with fulminant hepatitis. Hepatology 1998;28:1390 /7. [2] Fujiwara K. Annual report of research group for intractable liver disease, 2001. p. 24 /31. [3] Liang TJ, Hasegawa K, Rimon N, Wands J, Ben-Porath E. A hepatitis B virus mutant associated with an epidemic of fulminant hepatitis. N Engl J Med 1991;324:1705 /9. [4] Hasegawa K, Huang J, Wand JR, Obata H, Liang TJ. Association of hepatitis B viral precore mutations with fulminant hepatitis B in Japan. Virology 1991;185:460 /3. [5] Omata M, Ehata T, Yokosuka O, Hosoda K, Ohto M. Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis. N Engl J Med 1991;324:1699 /704. [6] Kosaka Y, Takase K, Kojima M, et al. Fulminant hepatitis B: induction by hepatitis B virus mutants defective in the precore region and incapable of encoding e antigen. Gastroenterology 1991;100:1087 /94. [7] Kojima M, Shimizu M, Tsuchimochi T, et al. Post-transfusion fulminant hepatitis B associated with precore-defective HBV mutants. Vox Sang 1991;60:34 /9. [8] Ehata T, Omata M, Chuang W, et al. Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis. N Engl J Med 1991;324:1699 /704. [9] Aye TT, Uchida T, Becker SO, Hirashima M. Variations of hepatitis B virus precore/core gene sequence in acute and fulminant hepatitis B. Dig Dis Sci 1994;6:1281 /7. [10] Chu CM, Yeh CT, Chiu CT, Sheen IS, Liaw YF. Precore mutant of hepatitis B virus prevails in acute and chronic infections in an area in which hepatitis B is endemic. J Clin Microbiol 1996;34:1815 /8. [11] Carman WF, Fagan EA, Hadziyannis S, et al. Association of a precore genomic variant of hepatitis B virus with fulminant hepatitis. Hepatology 1991;14:219 /22. [12] Sato S, Suzuki K, Akahane Y, et al. Hepatitis B virus strains with mutations in the core promoter in patients with fulminant hepatitis. Ann Intern Med 1995;122:241 /8. [13] Kaneko M, Uchida T, Moriyama M, et al. Probable implication of mutations of the X open reading frame in the onset of fulminant hepatitis B. J Med Virol 1995;47:204 /8. [14] Liang TJ, Hasegawa K, Munoz SJ, et al. Hepatitis B virus precore mutation and fulminant hepatitis in the United States. J Clin Invest 1994;93:550 /5.
124
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[15] Feray C, Gigou M, Samuel D, Bernuau J, Bismuth H, Brechot C. Low prevalence of precore mutations in hepatitis B virus DNA in fulminant hepatitis B in France. J Hepatol 1993;18:119 /22. [16] Laskus T, Persing DH, Nowicki MJ, Mosley MJ, Rakela J. Nucleotide sequence analysis of the precore region in patients with fulminant hepatitis B in the United States. Gastroenterology 1993;105:1173 /8. [17] Laskus T, Rakela J, Nowicki MJ, et al. Hepatitis B virus core promoter sequence analysis in fulminant and chronic hepatitis B. Gastroenterology 1995;109:1618 /23. [18] Sterneck M, Gunther S, Santantonio T, et al. Hepatitis B virus genomes of patients with fulminant hepatitis do not share a specific mutation. Hepatology 1996;24:300 /6. [19] Chan HL, Hussain M, Lok ASF. Different hepatitis B virus genotypes are associated with different mutations in the core promoter and precore regions during hepatitis B e antigen seroconversion. Hepatology 1999;29:976 /84. [20] Lindh M, Anderson A-S, Gusdal A. Genotypes, nt 1858 variants, and geographic origin of hepatitis B virus-large-scale analysis using a new genotyping method. J Infect Dis 1997;175:1285 /93. [21] Perrillo RP, Aach RD. The clinical course and chronic sequelae of hepatitis B virus infection. Semin Liver Dis 1981;1:15 /25.
[22] Ogawa M, Hasegawa K, Naritomi T, Torii N, Hayashi N. Clinical features and viral sequences of various genotypes of hepatitis B virus compared among patients with acute hepatitis B. Hepatol Res 2002;23:167 /77. [23] Kobayashi M, Arase Y, Ikeda K, et al. Clinical characteristics of patients infected with hepatitis B virus genotypes A, B, and C. J Gastroenterol 2002;37:35 /9. [24] Kao JH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology 2000;118:554 /9. [25] Orito E, Mizokami M, Sakugawa H, et al. A case /control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Hepatology 2001;33:218 /23. [26] Sugauchi F, Orito E, Ichida T, et al. Hepatitis B virus of genotype B with or without recombination with genotype C over the precore region plus the core gene. J Virol 2002;76:5985 /92. [27] Laras A, Koskinas J, Hadziyannis SJ. In vivo suppression of precore mRNA synthesis is associated with mutations in the hepatitis B virus core promoter. Virology 2002;295:86 /96. [28] Ono Y, Onda H, Sasada R, Igarashi K, Sugino Y, Nishioka K. The complete nucleotide sequences of the cloned hepatitis B virus DNA: subtype adr and adw. Nucl Acids Res 1983;11:1747 /57.
Genotypic analysis of hepatitis B virus from patients with fulminant hepatitis: comparison with acute self-limited hepatitis Riho Joh, Kiyoshi Hasegawa *, Miho Ogawa, Kayo Ishikawa, Aiko Iizuka, Takuma Naritomi, Naoko Kanai, Nobuyuki Torii, Etsuko Hashimoto, Naoaki Hayashi Department of Medicine, Institute of Gastroenterology, Tokyo Women’s Medical University, 8-1, Kawadacho, Shinjuku-ku, Tokyo 162-8666, Japan Received 20 August 2002; received in revised form 19 November 2002; accepted 16 January 2003
Abstract Aim/background: There is an increasing evidence that certain hepatitis B virus (HBV) strains may contribute to the pathogenesis of fulminant hepatitis B (FHB). Recently, we reported that genotypes of HBV influence the clinical course of acute self-limited hepatitis B (AHB). In this study, we compared clinical features of FHB between different HBV genotypes and compared the prevalence of each genotype between FHB and AHB patients. Methods: The subjects consisted of seven patients with FHB and 25 patients with AHB. The core promoter and precore region were directly sequenced following polymerase chain reaction, and genotype was determined by restriction fragment length polymorphism analysis of the S gene. Results: Of the seven FHB patients, one had genotype A, one had genotype B, four had genotype C, and one had genotype D. Six of the seven FHB patients were infected by heterosexual contact; one FHB patient who was not infected by heterosexual contact had genotype C. All four FHB patients with genotype C had a short duration clinical course. In one patient with genotype A, the time from onset of hepatitis to hepatic coma was 30 days. These results are similar to those of the patients with AHB, in which clinical course was longer in patients with genotype A than in patients with genotype C. Conclusion: Viral genotype can be used to predict the clinical course of both FHB and AHB. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Fulminant hepatitis; Genotype; Core promoter; Precore
1. Introduction Approximately 1% of patients infected with hepatitis B virus (HBV) develops fulminant hepatitis B (FHB), which is characterized by rapid destruction of the liver parenchyma [1]. In Japan, 34.7% of fulminant hepatitis is associated with HBV infection, including acute onset type and acute exacerbation of chronic hepatitis B (CHB) [2]. The survival rate of HBV-induced fulminant hepatitis is 50 and 30% in patients with acute-onset and chronic HBV infection, respectively. In other words, more than half of the patients with FHB ultimately requires liver transplantation or die of liver failure. The pathogenesis of FHB is not well understood. Previous studies have indicated that complicated interaction
* Corresponding author. Tel.: /81-3-3353-8111; fax: /81-3-52697435. E-mail address: [email protected] (K. Hasegawa).
between viral factors and host immune response may be involved. In previous studies, FHB was found to be associated with the precore stop codon mutation G1896A [3 /11] and two mutations in the core promoter region (A1762T and G1764A) [12,13]. These associations have mainly been found in countries with epidemic hepatitis B. In many reported cases of FHB in non-endemic areas, none of these three mutations was observed [14 /18]. Appearance of precore mutation is influenced by nt 1858, which is the counterpart of nt 1896 in the stemloop structure of pregenome RNA. The base at nt 1858 is cytosine (C) in genotypes A and F, and is thymidine (T) in genotypes B, C, D and E. Mutation from guanine (G) to adenine (A) at nt 1896 occurs readily in genotypes B, C, D and E possibly due to stabilization of the stemloop structure by the matching of adenine to thymidine [19]. In HBV of genotypes B and C, which are the predominant types in Asia, the base at nt 1858 tends to
1386-6346/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1386-6346(03)00011-1
A B C C C C D Heterosexual Heterosexual Heterosexual Unknown Heterosexual Heterosexual Heterosexual Death Death / / Death Death / / / / / / / / 30 5 7 7 7 8 5 /(14)//(41) /(0.9)//(95) /(4)//(84) /(0.9)//(96) /(3)//(51) /(3)//(84) /(1.0)//(91) 1 2 3 4 5 6 7
Clinical data were obtained on admission. a Days from onset to appearance of hepatic coma. b Route of infection.
41.0 8.3 7.1 10.7 4.6 7.9 3.8
527 3.480 4.910 5.008 4.850 8.130 11.060
18.4 12.5 26.0 17.7 16.9 B/10 24.7
/// /// /// /// /// /// ///
HBeAg/anti-HBe (COI) Table 1 Clinical features of seven patients with FHB
The subjects consisted of seven patients with FHB and 25 patients with acute self-limited hepatitis B (AHB). The seven patients with FHB (mean age, 419/ 19 years; four males and three females; Table 1) had been admitted to our hospital and were all diagnosed with acute-onset fulminant hepatitis. The criteria for FHB were development of hepatic encephalopathy, prolongation of the prothrombin time during the course of hepatitis and IgM anti-HBc antibody [21]. All FHB patients had no prior hepatitis. Three FHB patients survived to the end of the follow up period; all three recovered from hepatitis with loss of HBs antigen (HBsAg) and appearance of anti-HBs antibody in the follow up period. In all three surviving FHB patients, chronic hepatitis was excluded by histological examination. Four FHB patients died during the study period: three from liver failure and one (case 1) from multiple organ failure. In case 1, titer of anti-HBc antibody was high, because he was transferred from another hospital 21 days after onset and his anti-HBc antibody titer had not been checked at that hospital. He died after receiving a liver transplant from his father. Histological examination of his explanted liver showed acute hepatitis with submassive hepatic necrosis and no evidence of chronic hepatitis. Hepatitis B e antigen (HBeAg) was positive in three of the four patients who died, and negative in two of the three patients who survived. HBV DNA, which was measured by transcription mediated amplification (TMA), was undetectable in two patients: cases 1 and 2. Cases 1, 2, 6 and 7 were infected from unspecified sexual partners and cases 3 and 5 were infected from their spouses. The 25 AHB patients are described in detail elsewhere [22]. Briefly, we chose 25 patients, whose serum samples were available, from among 27 patients with acute hepatitis B treated at our hospital from 1996 to 2000. The AHB patient group consisted of 23 males and two females, ranging in age from 16 to 48 years (mean age, 329/8 years). AHB was diagnosed according to the following criteria: acute-onset liver dysfunction, IgM
IgM-HBc (LGE/ml)
2.1. Patients
23/M 70/M 28/F 61/M 34/F 49/M 22/F
2. Patients and methods
/3.7 /3.7 8.1 4.2 6.4 7.3 5.3
HBV DNA Daysa Brain edema Outcome Routeb
be T. In genotype A, which is common in European countries, the base at nt 1858 is C [20]. Therefore, it appears that such differences in prevalence of HBV genotype between geographic areas are related to differences in prevalence of the precore G1896A mutation. In this study, we examined viral factors influencing the pathogenesis of FHB by studying the genotype of HBV and the sequences of the core promoter and precore region.
2.7 3.3 3.1 3.1 2.8 3.2 3.2
Genotype
R. Joh et al. / Hepatology Research 26 (2003) 119 /124
Case age/sex T-Bil (mg/dl) ALT (IU/l) PT (%) HBsAg/anti-HBs
120
R. Joh et al. / Hepatology Research 26 (2003) 119 /124
anti-HbcAb-positive, no prior hepatitis, and recovery from hepatitis with loss of HBsAg and appearance of anti-HBs antibody in the follow-up period. 2.2. Determination of genotype The HBV genotype was determined by restriction fragment length polymorphism analysis after polymerase chain reaction (PCR-RFLP), as described by Lindh et al. [20]. Briefly, DNA extracted from patients’ sera was amplified by PCR using the following primers: 5?GTGGTGGACTTCTCTCAATTTTC-3? (sense; nt 256 /278) and 5?-AGGTAAAAAGGGACTCAAGAT3? (anti-sense; nt 796/776). Then, 5 ml of PCR product was mixed with five units of TSP509i, 2 ml of 10 / buffer and 12 ml of water, and then incubated at 65 8C for 3 h. In a separate reaction, 5 ml of PCR product was mixed with five units of HinfI, 2 ml of 10/ buffer and 12 ml of water, and then incubated at 37 8C for 3 h. These mixtures were then resolved on 3% agarose gels and stained with ethidium bromide. The restriction profiles were visually interpreted. 2.3. Amplification of HBV DNA by PCR and sequencing of PCR products Serum (50 ml) was diluted with 50 ml of sterile distilled water and mixed with 100 ml of a mixture containing 25 mM sodium acetate, 2.5 mM EDTA (pH 8.0), 1% SDS, 2 mg/ml of proteinase K and 10 mg/ml of yeast t-RNA as a carrier. Following digestion at 65 8C for 2 h, viral nucleic acids were extracted twice with phenol /chloroform, extracted once with chloroform, ethanol-precipitated and suspended in water. The core promoter and precore regions, consisting of a 1359-bp sequence extending from nt 1435 to 2793, were amplified by PCR using the extracted HBV DNA. The primers were 5?-CGTCGGCGCTGAATCC-3? (sense; nt 1435/1450) and 5?-CGAGGCGAGGGAGTTCTTCTTC-3? (antisense; nt 2793/2771). Then, 5 ml of the extracted DNA was mixed with 45 ml of PCR mixture containing 400 nM of the primers, and amplified by 35 cycles of 94 8C for 1 min, 55 8C for 1 min and 72 8C for 3 min, in an automatic DNA thermal cycler (Perkin/Elmer Cetus, Norwalk, CT). The PCR products were directly sequenced by dideoxy-chain termination using an AutoRead DNA sequencing kit (Pharmacia LKB Biotechnology, Uppsala, Sweden) with the anti-sense primer used in PCR.
3. Results Of the seven FHB patients, one had genotype A, one had genotype B, four had genotype C and one had genotype D. In patients 1 and 2, who had genotypes A
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and B, respectively, HBV DNA was not detectable by TMA. In contrast, the four patients with genotype C had HBV DNA levels ranging from 4.2 to 8.1 LGE/ml (mean 6.59/1.7 LGE/ml). Patient 1, who had genotype A, had a long-duration clinical course of hepatitis: 30 days from onset to coma. This patient had high total bilirubin and low peak ALT. However, he resisted intensive treatment, including artificial liver support, and eventually died due to complications after liver transplantation. With the exception of case 1, there was no difference in days from onset to coma between patients with genotypes B, C and D. Three patients who died (one patient with genotype B and two with genotype C) exhibited brain edema. Sequence analysis of core promoter and precore region revealed double mutation at nt 1762 and 1764 in all four patients with genotype C, whereas no patients with genotype A, B or D had this double mutation (Fig. 1). This double mutation was detected in four of the 25 AHB patients (Table 2). Only two of the six AHB patients with genotype C had the double mutation at nt 1762 and 1764 (Table 2). No other common mutation in the core promoter region was found in the FHB patients. Thus, mutations in the core promoter region were more prevalent in the FHB patients than in the AHB patients, but this difference was not significant. In the precore region, nt 1858 was thymidine in all four FHB patients with genotype C and the one FHB patient with genotype D. Of these five patients with thymidine at 1858, four had a G-to-A mutation at 1896. Two patients, one with genotype A and one with genotype B, had cytidine at 1858 and guanine at 1896. Only two of the seven AHB patients with thymidine at 1858 had a Gto-A mutation at 1896.
4. Discussion In this study, four of the seven FHB patients had HBV of genotype C and all patients with genotype C had a short course of fulminant hepatitis. Previously, we reported that genotype A was most prevalent in patients with AHB in our hospital, and that the clinical course of acute hepatitis caused by genotype A tended to progress slowly [22]. In this FHB patient with genotype A, the inability to detect HBV DNA by the TMA method on admission may be due to the long interval between onset and admission. Together, these findings suggest the possibility that, in patients with genotype A, weak capacity for recovery from liver injury contributes to the development of subacute fulminant hepatitis. In contrast, acute hepatitis caused by HBV of genotype C was characterized by a shorter course and more severe hepatic injury than acute hepatitis caused by HBV of other genotypes. In this study, the one fulminant hepatitis patient with genotype A had a slowly progres-
R. Joh et al. / Hepatology Research 26 (2003) 119 /124
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Fig. 1. Upper part: nucleotide sequences of a portion of core promoter region of HBV in seven Japanese patients with fulminant hepatitis, and relation to HBeAg/HBeAb phenotype. Consensus sequences adr and adw are described elsewhere [28]. Letters surrounded by open squares in consensus sequences represent A at 1762 and G at 1764. Lower part: nucleotide sequences of precore region. Letters surrounded by open squares in consensus sequences represent T at 1858 and G at 1896.
sing course, developing hepatic coma 30 days after appearance of the first symptom, which was general malaise. The reason for differences in clinical course of acute and fulminant hepatitis between genotypes is unclear. One explanation is that HBV of genotype A has cytidine at nt 1858, which is the counterpart of nt
1896 in the stem-loop structure of pregenome RNA, and that this inhibits nucleotide substitution from guanine to adenine at nt 1896, in turn inhibiting termination of HBeAg production. In fact, patient 1 had cytosine at nt 1858 and guanine at nt 1896. In patients with CHB, genotype A is significantly more likely to be associated
Table 2 Comparison of sequences of core promoter and precore region between patients with AHB and fulminant hepatitis Genotype (n )
HBeAg
Core promoter (1762/1764)
Precore (1858)
Precore (1896)
/
/
A/G
T/A
ND
T
C
G
A
AHB
A (14) B (5) C (6)
12 4 5
2 1 1
9 4 4
2 0 2
3 1 0
0 4 3
14 1 3
14 5 4
0 0 2
FHB
A (1) B (1) C (4) D (1)
1 0 3 0
0 1 1 1
1 1 0 1
0 0 4 0
0 0 0 0
0 0 4 1
1 1 0 0
1 1 1 0
0 0 3 1
ND: not detectable.
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with HBeAg-positive phenotype at first examination than other genotypes [23]. Thus, continuous HBeAgpositive phenotype may contribute to long duration of liver injury. The other explanation is that severe acute hepatitis with long clinical course can be controlled by antiviral agents such as lamivudine or interferon, if the therapy is started in the early phase of hepatitis. However, once acute fulminant hepatitis has developed, it is difficult to prevent uncontrolled liver failure. It has been reported that hepatitis of genotype B has a greater tendency to subside over time and is more easily controlled by medication than hepatitis of genotype A or C [24,25]. However, it has been reported that some patients with genotype B have rapidly progressive and severe clinical courses [26]. In these latter cases, part of HBV genotype B was substituted with sequences of genotype C by recombination. This type of genotype B HBV, which is epidemic in southeast Asia and rare in Japan, is called genotype Ba [26]. Based on results of sequence analysis, we classified the HBV of case 2 as genotype BJ (data not shown). It is unclear what relationships exist between subclassification of genotype B and clinical course of acute onset hepatitis B. The present FHB patients had more mutations in both the core promoter region and precore region than AHB patients. The mutations A1762T and G1764A are reportedly associated with fulminant hepatitis in Japan [12]. However, in case 1 (genotype A) and case 2 (genotype B), the nt at 1762 and 1764 were A and G, respectively. Together, genotypes A and B constitute a minority of HBV cases in Japan. Recent studies in the United States and Germany indicate that the A1762T/ G1764A double mutation is not associated with FHB [17,18]. Thus, there appear to be differences between Caucasian and Asian patients in the significance of various sequences in the core promoter region. Recently, it has been reported that double mutations in the core promoter region contribute to enhanced replication of HBV and active liver diseases [27]. In this study, mutations in the core promoter region were only detected in FHB patients with genotype C. However, the number of patients with fulminant hepatitis was too small to determine associations between core promoter sequences, HBV genotype and development of fulminant hepatitis. Therefore, a study with a larger number of FHB patients is needed. In this study, guanine at nt 1896 was converted to adenine in four of the five FHB patients with thymidine at nt 1858. In contrast, G-to-A mutation at nt 1896 was detected in only two of the seven AHB patients with thymidine at nt 1858. These results suggest that G-to-A mutation at nt 1896, which produces a stop codon in the precore region, results from active replication, which is observed in patients with fulminant hepatitis. Cases 2 and 3 (both of which had cytidine at nt 1858) had guanine at nt 1896. Previously, wild type sequence at nt
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1896 has reportedly been detected in American patients with fulminant hepatitis [14]. Although genotype was not analyzed in that study, we can speculate that differences in HBV genotype distribution between American and Japanese hepatitis patients are responsible for these differences in the frequencies of different sequences at nt 1896 between Japanese and American studies. In conclusion, HBV genotype can be used to predict the clinical course of acute onset hepatitis. However, only a limited number of patients with fulminant hepatitis were examined in this study. A larger study may produce more practical information for use in diagnosis and therapy of acute-onset hepatitis B.
References [1] Sterneck M, Kalinina T, Gu¨nter S, et al. Functional analysis of HBV genomes from patients with fulminant hepatitis. Hepatology 1998;28:1390 /7. [2] Fujiwara K. Annual report of research group for intractable liver disease, 2001. p. 24 /31. [3] Liang TJ, Hasegawa K, Rimon N, Wands J, Ben-Porath E. A hepatitis B virus mutant associated with an epidemic of fulminant hepatitis. N Engl J Med 1991;324:1705 /9. [4] Hasegawa K, Huang J, Wand JR, Obata H, Liang TJ. Association of hepatitis B viral precore mutations with fulminant hepatitis B in Japan. Virology 1991;185:460 /3. [5] Omata M, Ehata T, Yokosuka O, Hosoda K, Ohto M. Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis. N Engl J Med 1991;324:1699 /704. [6] Kosaka Y, Takase K, Kojima M, et al. Fulminant hepatitis B: induction by hepatitis B virus mutants defective in the precore region and incapable of encoding e antigen. Gastroenterology 1991;100:1087 /94. [7] Kojima M, Shimizu M, Tsuchimochi T, et al. Post-transfusion fulminant hepatitis B associated with precore-defective HBV mutants. Vox Sang 1991;60:34 /9. [8] Ehata T, Omata M, Chuang W, et al. Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis. N Engl J Med 1991;324:1699 /704. [9] Aye TT, Uchida T, Becker SO, Hirashima M. Variations of hepatitis B virus precore/core gene sequence in acute and fulminant hepatitis B. Dig Dis Sci 1994;6:1281 /7. [10] Chu CM, Yeh CT, Chiu CT, Sheen IS, Liaw YF. Precore mutant of hepatitis B virus prevails in acute and chronic infections in an area in which hepatitis B is endemic. J Clin Microbiol 1996;34:1815 /8. [11] Carman WF, Fagan EA, Hadziyannis S, et al. Association of a precore genomic variant of hepatitis B virus with fulminant hepatitis. Hepatology 1991;14:219 /22. [12] Sato S, Suzuki K, Akahane Y, et al. Hepatitis B virus strains with mutations in the core promoter in patients with fulminant hepatitis. Ann Intern Med 1995;122:241 /8. [13] Kaneko M, Uchida T, Moriyama M, et al. Probable implication of mutations of the X open reading frame in the onset of fulminant hepatitis B. J Med Virol 1995;47:204 /8. [14] Liang TJ, Hasegawa K, Munoz SJ, et al. Hepatitis B virus precore mutation and fulminant hepatitis in the United States. J Clin Invest 1994;93:550 /5.
124
R. Joh et al. / Hepatology Research 26 (2003) 119 /124
[15] Feray C, Gigou M, Samuel D, Bernuau J, Bismuth H, Brechot C. Low prevalence of precore mutations in hepatitis B virus DNA in fulminant hepatitis B in France. J Hepatol 1993;18:119 /22. [16] Laskus T, Persing DH, Nowicki MJ, Mosley MJ, Rakela J. Nucleotide sequence analysis of the precore region in patients with fulminant hepatitis B in the United States. Gastroenterology 1993;105:1173 /8. [17] Laskus T, Rakela J, Nowicki MJ, et al. Hepatitis B virus core promoter sequence analysis in fulminant and chronic hepatitis B. Gastroenterology 1995;109:1618 /23. [18] Sterneck M, Gunther S, Santantonio T, et al. Hepatitis B virus genomes of patients with fulminant hepatitis do not share a specific mutation. Hepatology 1996;24:300 /6. [19] Chan HL, Hussain M, Lok ASF. Different hepatitis B virus genotypes are associated with different mutations in the core promoter and precore regions during hepatitis B e antigen seroconversion. Hepatology 1999;29:976 /84. [20] Lindh M, Anderson A-S, Gusdal A. Genotypes, nt 1858 variants, and geographic origin of hepatitis B virus-large-scale analysis using a new genotyping method. J Infect Dis 1997;175:1285 /93. [21] Perrillo RP, Aach RD. The clinical course and chronic sequelae of hepatitis B virus infection. Semin Liver Dis 1981;1:15 /25.
[22] Ogawa M, Hasegawa K, Naritomi T, Torii N, Hayashi N. Clinical features and viral sequences of various genotypes of hepatitis B virus compared among patients with acute hepatitis B. Hepatol Res 2002;23:167 /77. [23] Kobayashi M, Arase Y, Ikeda K, et al. Clinical characteristics of patients infected with hepatitis B virus genotypes A, B, and C. J Gastroenterol 2002;37:35 /9. [24] Kao JH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology 2000;118:554 /9. [25] Orito E, Mizokami M, Sakugawa H, et al. A case /control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Hepatology 2001;33:218 /23. [26] Sugauchi F, Orito E, Ichida T, et al. Hepatitis B virus of genotype B with or without recombination with genotype C over the precore region plus the core gene. J Virol 2002;76:5985 /92. [27] Laras A, Koskinas J, Hadziyannis SJ. In vivo suppression of precore mRNA synthesis is associated with mutations in the hepatitis B virus core promoter. Virology 2002;295:86 /96. [28] Ono Y, Onda H, Sasada R, Igarashi K, Sugino Y, Nishioka K. The complete nucleotide sequences of the cloned hepatitis B virus DNA: subtype adr and adw. Nucl Acids Res 1983;11:1747 /57.