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HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2001 AASLD ABSTRACTS 615A 1771 GENOTYPE ANALYSIS OF HEPATITIS B VIRUS IN TURKISH POPULA- TION USING RESTRICTION FRAGMENT LENGTH POLYMORPHISM PATTERNS. Pinar Uysal, Marmara University, School of Medicine, Medical Biology Dept, Istanbul Turkey; Naz Oguzoglu, Haydarpasa Numune Hospital, Istanbul Turkey; Ayse Ozer, Marmara University, School of Medicine, Medical Biology Dept, Istanbul Turkey Six different HBV genotypes have been defined comparing the complete HBV genome. The distribution of genotypes among infected populations varies geo- graphically and it has been proposed that there is a relationship between HBV genotypes and development of chronic liver disease. This study is therefore designed for the classification of HBV genotypes in Turkish population using restriction fragment length polymorphism (RFLP) analysis of the S gene re- gion. HBV DNA extracted from forty-eight serum samples were subjected to polymerase chain reaction (PCR) using primers from the S region of HBV genome. Restriction enzymes, Hph I (Genotype F), Nci I (Genotype E), Alw I (Genotype C), Ear I (Genotype B), and Nla IV (Genotype A and D), were used for RFLP analysis of amplified product of HBV DNA. In the 48 serum samples investigated, 3 different genotypes were detected. These are as follows; Geno- type D: 41 (85.41%), Genotype A: 5(10.41%), and Genotype F: 2 (4.16%). This sLudy shows a molecular heterogeneity of HBV in the Turkish population with at least 3 different genotypes observed in the 48 samples studied. The genotype El is by far the most common genotype in the Turkish population as is the case in all other Mediterranean countries. 1772 TUMOR NECROSIS FACTOR-ALPHA (TNF-c0 PROMOTER POLYMOR- PHISM IN VARIOUS STATUS OF HEPATITIS B VIRUS (HBV) INFEC- TION. Song Kun Hoon, Han Kwang-Hyub, Cheong Jae Youn, Shin Jeon Soo, Kim Jeong Hee, Lee Ji Sook, Chon Chae Yoon, Moon Young Myoung, Yonsei University College of Medicine, Seoul South Korea Backgrounds: The reason for persistence of HBV are still poorly understood. TNF-cz has been known to abolish HBV gene expression and replication, Re- cently, two G to A transitions in the promoter region at position -308 & -238 have been shown to influence TNF-c~expression. Additionally HLA-DRBI*13 has been reported to be related to the clearance of HBV. The aim of this study was to determine whether TNF-c~ promoter polymorphism has a special rela- tionship with outcome of HBV infection, Patients and Methods: A total of 68 patients were classified into four groups: 1) control group [N=10, HLA- DRBI*13 (-)ve, HBsAg, Anti-HBc, Anti-HBs all (-)ve], 2) clearance group [N=22, either HLA-DRBI*13 (+)re or (-)ve, HBsAg (-)ve, Anti-HBc and Anti- HBs (+)ve], 3) HBeAg-negative carrier group [N = 13, HBsAg (+)ve, HBeAg (-)ve, showing normal transaminase level], 4) progression group IN = 23, HB- sAg (+)ve, chronic hepatitis, cirrhosis or HCC] The TNF-c~ promoter poly- morphism was tested by ABI Prism®SnaPshot TM ddNTP Primer Extension Kit (primers; -308 sense: 5' AATAG GTTTT GAGGG GCATG 3', -308 antisense: 5' TTTTT TCTGG AGGCT GAACC CCGTC C 3', -238 sense: 5' TTTTT TTTTT TT2"TT TAGAA GACCC CCCTC GGAAT C 3', -238 antisense: 5' TTT TTTTT TTTTT TTTTT TTTTT ACTCC CCATC CTCCC TGCTC 3') Results: TNF308.2 (G to A transition) was observed in 7/22(31.8%) patients in clear- ance group, which was significantly more frequent compared with control group (0/10, 0%), HBeAg-negative carrier group (1/13, 7.7%) and progression group (3/23, 13%)(p=0.0388). Substantial genetic association was also noted between TNF308.2 and HLA-DRBI*13 (TNE308.2 & DRBI*13 positive: 58.3% vs. TNF308.2 & DRBI*13 negative: O%)(p<O.O01) The frequency of TNF-~ -238 polymorphism was not different among the four groups. Conclu- sion: These findings suggested an association between the TNF-c¢ promoter polymorphism at position -308 and the clearance of HBV infection. But sub- stantial genetic association was also suspected between TNF 308.2 and HLA- DRBI* 13. Therefore further study is needed to clarify the relationship between HLA-DRBI*13 and the clearance of HBV infection. 1773 LONG-TERM FOLLOW-UP AFTER CLEARANCE OF SERUM HBSAG IN CHRONIC HEPATITIS B VIRUS CARRIERS. Manuel Rodriguez, Araneha Alvarez, Monica G Espiga, Mercedes Rodriguez, Nieves G Sotorrio, Luisa G Dieguez, Ramon Perez, Lnis Rodrigo, Hospital Central de Asturias, Oviedo Spain BACKGROUND: Delayed HBsAg sero-clearance in chronic hepatitis B virus (HBV) carriers is an unusual event and it has been considered the final point in tire resolutive process of chronic HBV infection. However, the prognosis of patients who clear HBsAg is controversial. AIM: To determine the clinical and serological evolution after HBsAgclearance in a group of chronic HBV carriers. METHODS: During a mean follow-up period of 75 + 41 months, 67 out of 937 chronic HBV carriers cleared HBsAg (absence of HBsAg in at least two consec- utive samples obtained one month apart). Five patients with HCV and/or HDV infection and 3 other patients who had already had liver complications before HBsAg clearance were excluded from the analysis. Of the remaining 59 pa- t:Lents, 37 were inactive chronic HBV carriers and 22 had liver disease (17 chronic hepatitis and 5 cirrhosis). Twelve patients had been treated with in- terferon-alfa. Patients were followed at different intervals during 62 -+ 32 (12-159) months after HBsAg clearance. RESULTS: HBsAg reappeared in 2 patients (1 with HIV infection and i with Down's syndrome). The cumulative probability of HBsAg reappearance at 3, 5 and 7 years was 0, 3 and 9% respec- tively. 43/59 (73%) patients developed antiHBs at levels --> 10 mIU/ml (geo- metric mean titer: 294-+327 mIU/ml). During the follow-up, the ALT levels remained within the normal range in 57/59 (96.6%) patients. Only one patient, in whom HBsAg had reappeared, developed liver complications and died from hver disease. None of the 59 patients developed hepatocellular carcinoma. A hver biopsy was performed in 10 patients after HBsAg clearance and a decreas- ing in the necroinflammatory component of the HAI was observed when com- pared with the previous biopsy (1.2 + 1.6 vs 8.4 -+ 4.1; p<0.001); HBsAg staining in the liver tissue was negative in all cases. CONCLUSIONS: HBsAg clearance in chronic HBV carriers is an irreversible process except under im- munosuppessive circumstances. The clinical evolution after HBsAg clearance is excelent in patients without previous liver complications and who are not infected with other hepatotrophic viruses. 1774 THE CLINICAL SIGNIFICANCE OF HBV GENOTYPES IN JAPAN AND CHINA. Miki Kaneko, Yasuo Nagamatu, Nihon University School of Japan, Tokyo Japan; Seiji Okayama, Hiroaki Yamagami, Nihon University School of Medicine, Tokyo Japan; Atuo Shioda, Mituhiko Moriyama, Yasuynki Arakawa, Nihon University School of Japan, Tokyo Japan Background and Aim: The clinical condition of patients infected with hepatitis type B virus (HBV) and the response of these patients to IFN therapy has been reported to vary depending upon the genotypes of HBV. In this study, we collected serum samples from HBe antigen-positive patients with chronic hep- atitis type B in Japan (Tokyo and Miyakojima in Okinawa) and China (Yan- bian), and determined the genotypes of HBV in these samples using ELISA. In addition, we have examined whether there are any differences in laboratory findings and in histopathotogical changes in the liver among patients infected with different genotypes. Moreover, we drew genetic dendrograms on the basis of genetic maps in some patients, and examined the distance between genes in identical genotypes. Methods: The subjects included 50 patients in Miya- kojima, Okinawa, 10 patients in Yanbian, China and 66 patients in Tokyo that were diagnosed with chronic hepatitis type B. Blood samples taken from all of the Japanese patients were found to be positive for HBe antigens. HBV geno- types were determined using ELISA, in which monoclonal antibodies (HBV genotype EIA, Toknshu-meneki Institute, Japan) against the genotype-specific epitopes located in the pre-S2 region were used. Histopathological changes in the liver were evaluated with respect to the following 5 items: Periportal (1), parenchymal (2), portal (3), portal lymphoid reactions (4) and irregular re- generation (IR, 5). The intensities of inflammatory changes in items 1-4 and the degree of IR were rated and given scores from 0 (none) to 4 (severe). The degree of intrahepatic fibrosis was expressed by the F score. Results: No sig- nificant differences in age and sex were observed among patients in the 3 districts. In Miyakojima, genotypes B and C were found in 48 cases (96%) and 2 cases (4%), respectively. In Yanbian, all cases were found to consist of geno- type C. In Tokyo, genotypes A, B and C were found in 3 (4.5%), 6 (9%) and 57 cases (86.5%), respectively, among the 66 cases diagnosed with chronic hep- atitis. In HBe antigen-positive patients with chronic hepatitis type B who un- derwent a liver biopsy, no significant differences were found among the differ- ent genotypes in the intensity ofinfiammatory cell infiltration and in the degree of IR in the liver. Conclusions: Our results indicate that HBV genotype C was dominant in Tokyo and China (Yanbian), whereas genotype B was dominant in Okinawa (Miyakojtma), which is in East Asia as are the other 2 districts. In addition, our results suggest that no differences exist between the pathogenesis of genotypes B and C of HBV.
Transcript of document
HEPATOLOGY Vol. 34, No. 4, Pt. 2, 2001 AASLD ABSTRACTS 615A
1771
GENOTYPE ANALYSIS OF HEPATITIS B VIRUS IN TURKISH POPULA- TION USING RESTRICTION FRAGMENT LENGTH POLYMORPHISM PATTERNS. Pinar Uysal, Marmara University, School of Medicine, Medical Biology Dept, Istanbul Turkey; Naz Oguzoglu, Haydarpasa Numune Hospital, Istanbul Turkey; Ayse Ozer, Marmara University, School of Medicine, Medical Biology Dept, Istanbul Turkey
Six different HBV genotypes have been defined comparing the complete HBV genome. The distribution of genotypes among infected populations varies geo- graphically and it has been proposed that there is a relationship between HBV genotypes and development of chronic liver disease. This study is therefore designed for the classification of HBV genotypes in Turkish population using restriction fragment length polymorphism (RFLP) analysis of the S gene re- gion. HBV DNA extracted from forty-eight serum samples were subjected to polymerase chain reaction (PCR) using primers from the S region of HBV genome. Restriction enzymes, Hph I (Genotype F), Nci I (Genotype E), Alw I (Genotype C), Ear I (Genotype B), and Nla IV (Genotype A and D), were used for RFLP analysis of amplified product of HBV DNA. In the 48 serum samples investigated, 3 different genotypes were detected. These are as follows; Geno- type D: 41 (85.41%), Genotype A: 5(10.41%), and Genotype F: 2 (4.16%). This sLudy shows a molecular heterogeneity of HBV in the Turkish population with at least 3 different genotypes observed in the 48 samples studied. The genotype El is by far the most common genotype in the Turkish population as is the case in all other Mediterranean countries.
1772
TUMOR NECROSIS FACTOR-ALPHA (TNF-c0 PROMOTER POLYMOR- PHISM IN VARIOUS STATUS OF HEPATITIS B VIRUS (HBV) INFEC- TION. Song Kun Hoon, Han Kwang-Hyub, Cheong Jae Youn, Shin Jeon Soo, Kim Jeong Hee, Lee Ji Sook, Chon Chae Yoon, Moon Young Myoung, Yonsei University College of Medicine, Seoul South Korea
Backgrounds: The reason for persistence of HBV are still poorly understood. TNF-cz has been known to abolish HBV gene expression and replication, Re- cently, two G to A transitions in the promoter region at position -308 & -238 have been shown to influence TNF-c~ expression. Additionally HLA-DRBI* 13 has been reported to be related to the clearance of HBV. The aim of this study was to determine whether TNF-c~ promoter polymorphism has a special rela- tionship with outcome of HBV infection, Patients and Methods: A total of 68 patients were classified into four groups: 1) control group [N=10, HLA- DRBI*13 (-)ve, HBsAg, Anti-HBc, Anti-HBs all (-)ve], 2) clearance group [N=22, either HLA-DRBI* 13 (+)re or (-)ve, HBsAg (-)ve, Anti-HBc and Anti- HBs (+)ve], 3) HBeAg-negative carrier group [N = 13, HBsAg (+)ve, HBeAg (-)ve, showing normal transaminase level], 4) progression group IN = 23, HB- sAg (+)ve, chronic hepatitis, cirrhosis or HCC] The TNF-c~ promoter poly- morphism was tested by ABI Prism ® SnaPshot TM ddNTP Primer Extension Kit (primers; -308 sense: 5' AATAG GTTTT GAGGG GCATG 3', -308 antisense: 5' TTTTT TCTGG AGGCT GAACC CCGTC C 3', -238 sense: 5' TTTTT TTTTT TT2"TT TAGAA GACCC CCCTC GGAAT C 3', -238 antisense: 5' TTT TTTTT TTTTT TTTTT TTTTT ACTCC CCATC CTCCC TGCTC 3') Results: TNF308.2 (G to A transition) was observed in 7/22(31.8%) patients in clear- ance group, which was significantly more frequent compared with control group (0/10, 0%), HBeAg-negative carrier group (1/13, 7.7%) and progression group (3/23, 13%)(p=0.0388). Substantial genetic association was also noted between TNF308.2 and HLA-DRBI*13 (TNE308.2 & DRBI*13 positive: 58.3% vs. TNF308.2 & DRBI*13 negative: O%)(p<O.O01) The frequency of TNF-~ -238 polymorphism was not different among the four groups. Conclu- sion: These findings suggested an association between the TNF-c¢ promoter polymorphism at position -308 and the clearance of HBV infection. But sub- stantial genetic association was also suspected between TNF 308.2 and HLA- DRBI* 13. Therefore further study is needed to clarify the relationship between HLA-DRBI* 13 and the clearance of HBV infection.
1773
LONG-TERM FOLLOW-UP AFTER CLEARANCE OF SERUM HBSAG IN CHRONIC HEPATITIS B VIRUS CARRIERS. Manuel Rodriguez, Araneha Alvarez, Monica G Espiga, Mercedes Rodriguez, Nieves G Sotorrio, Luisa G Dieguez, Ramon Perez, Lnis Rodrigo, Hospital Central de Asturias, Oviedo Spain
BACKGROUND: Delayed HBsAg sero-clearance in chronic hepatitis B virus (HBV) carriers is an unusual event and it has been considered the final point in tire resolutive process of chronic HBV infection. However, the prognosis of patients who clear HBsAg is controversial. AIM: To determine the clinical and serological evolution after HBsAg clearance in a group of chronic HBV carriers. METHODS: During a mean follow-up period of 75 + 41 months, 67 out of 937 chronic HBV carriers cleared HBsAg (absence of HBsAg in at least two consec- utive samples obtained one month apart). Five patients with HCV and/or HDV infection and 3 other patients who had already had liver complications before HBsAg clearance were excluded from the analysis. Of the remaining 59 pa- t:Lents, 37 were inactive chronic HBV carriers and 22 had liver disease (17 chronic hepatitis and 5 cirrhosis). Twelve patients had been treated with in- terferon-alfa. Patients were followed at different intervals during 62 -+ 32 (12-159) months after HBsAg clearance. RESULTS: HBsAg reappeared in 2 patients (1 with HIV infection and i with Down's syndrome). The cumulative probability of HBsAg reappearance at 3, 5 and 7 years was 0, 3 and 9% respec- tively. 43/59 (73%) patients developed antiHBs at levels --> 10 mIU/ml (geo- metric mean titer: 294-+327 mIU/ml). During the follow-up, the ALT levels remained within the normal range in 57/59 (96.6%) patients. Only one patient, in whom HBsAg had reappeared, developed liver complications and died from hver disease. None of the 59 patients developed hepatocellular carcinoma. A hver biopsy was performed in 10 patients after HBsAg clearance and a decreas- ing in the necroinflammatory component of the HAI was observed when com- pared with the previous biopsy (1.2 + 1.6 vs 8.4 -+ 4.1; p<0.001); HBsAg staining in the liver tissue was negative in all cases. CONCLUSIONS: HBsAg clearance in chronic HBV carriers is an irreversible process except under im- munosuppessive circumstances. The clinical evolution after HBsAg clearance is excelent in patients without previous liver complications and who are not infected with other hepatotrophic viruses.
1774
THE CLINICAL SIGNIFICANCE OF HBV GENOTYPES IN JAPAN AND CHINA. Miki Kaneko, Yasuo Nagamatu, Nihon University School of Japan, Tokyo Japan; Seiji Okayama, Hiroaki Yamagami, Nihon University School of Medicine, Tokyo Japan; Atuo Shioda, Mituhiko Moriyama, Yasuynki Arakawa, Nihon University School of Japan, Tokyo Japan
Background and Aim: The clinical condition of patients infected with hepatitis type B virus (HBV) and the response of these patients to IFN therapy has been reported to vary depending upon the genotypes of HBV. In this study, we collected serum samples from HBe antigen-positive patients with chronic hep- atitis type B in Japan (Tokyo and Miyakojima in Okinawa) and China (Yan- bian), and determined the genotypes of HBV in these samples using ELISA. In addition, we have examined whether there are any differences in laboratory findings and in histopathotogical changes in the liver among patients infected with different genotypes. Moreover, we drew genetic dendrograms on the basis of genetic maps in some patients, and examined the distance between genes in identical genotypes. Methods: The subjects included 50 patients in Miya- kojima, Okinawa, 10 patients in Yanbian, China and 66 patients in Tokyo that were diagnosed with chronic hepatitis type B. Blood samples taken from all of the Japanese patients were found to be positive for HBe antigens. HBV geno- types were determined using ELISA, in which monoclonal antibodies (HBV genotype EIA, Toknshu-meneki Institute, Japan) against the genotype-specific epitopes located in the pre-S2 region were used. Histopathological changes in the liver were evaluated with respect to the following 5 items: Periportal (1), parenchymal (2), portal (3), portal lymphoid reactions (4) and irregular re- generation (IR, 5). The intensities of inflammatory changes in items 1-4 and the degree of IR were rated and given scores from 0 (none) to 4 (severe). The degree of intrahepatic fibrosis was expressed by the F score. Results: No sig- nificant differences in age and sex were observed among patients in the 3 districts. In Miyakojima, genotypes B and C were found in 48 cases (96%) and 2 cases (4%), respectively. In Yanbian, all cases were found to consist of geno- type C. In Tokyo, genotypes A, B and C were found in 3 (4.5%), 6 (9%) and 57 cases (86.5%), respectively, among the 66 cases diagnosed with chronic hep- atitis. In HBe antigen-positive patients with chronic hepatitis type B who un- derwent a liver biopsy, no significant differences were found among the differ- ent genotypes in the intensity ofinfiammatory cell infiltration and in the degree of IR in the liver. Conclusions: Our results indicate that HBV genotype C was dominant in Tokyo and China (Yanbian), whereas genotype B was dominant in Okinawa (Miyakojtma), which is in East Asia as are the other 2 districts. In addition, our results suggest that no differences exist between the pathogenesis of genotypes B and C of HBV.
