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Hepatitis B virus code promoter sequence analysis in fulminant adn chronic hepatitis B
260A
613
AASLD ABSTRACTS
PRESERVATION OF CO2-DEPENDENT REGULATION OF CEREBRAL ARTERIAL BLOOD FLOW VELOCITY IN END STAGE LIVER DISEASE. JJ Katz. MS Mandell, B Barton, RM House. BM Bilir. GT Eversan. S Zamudin. Depts. of Anesth., Psychiatry, Medicine, University of Colorado School of Medicine, Denver.
614 HBV CORE PROMOTOR MUTATIONS CAUSE ENHANCED VIRAL REPLICATION IN ASSOCIATION WITH FULMINANT HEPATITIS. TF Baumert. SA Roonrs. K Haseaewa. and TJ Liarm. GI Unit, Dept of Medicine, Mass General Hosp and Harvard Medical School, Boston, MA. Mutations in hepatitis B virus (HBV) have been associated with the development of tulminant hepatitis. However, very few studies have addressed the functional relevance of these mutations. We recently cloned, sequenced, and characterized functionally the viral genome of a HBV strain associated with an epidemic of fulminant hepatitis (FH strain). Besides the well described precore mutations at nucleotide (nt) positions 1898 and 1901, numerous other mutations in various regions of the HBV genome were found. This HBV strain was functionally characterized by an increased encapsidation of pregenomic RNA leading to highly enhanced replication, which was not a result of the above mentioned precore mutations. We mapped the mutation(s) responsible for the enhanced replication of the FH strain to the core promotor. In order to define the responsible mutations, we introduced various mutations of the FH strain into a wild-type construct by site-directed mutagenesis. Viral antigen synthesis, transcription, encapsidation of pregenomic viral RNA, and replication of these mutant constructs were analyzed in comparison with those of wild-type strains. Analysis of viral replication showed that only the combined mutations of C to T at nt 1768 and T to A at nt 1770 directed the production of high level of coreassociated replicative intermediates. Similar to the FH strain, this mutant also displayed an enhanced encapsidation of pregenomic RNA. Cells transfected with wild-type and mutant HBV DNAs showed similar levels of total viral RNA and viral proteins, indicating that transcription and protein expression is not affected significantly by the introduced mutations; To extend our findings, we sequenced the core promotor region of six other viral strains from patients with fulminant hepatitis. Five out of six viral strains contained either the above mentioned or other mutations in the core promotor. Conclusion: Our studies show that two adjacent mutations in the core promolor lead to enhanced viral replication on the basis of increased encapsidation. Furthermore, functional characterization of the core promotor mutant suggests that mutations in this genomic region of HBV may play an important role in the pathogenesis of fulminant hepatic failure.
Although portal shunting and humoral mediators are implicated in portal-systemic encephalopathy, other factors, such as impaired astoregulation of cerebral blood flow (CBF) may contribute. We asked whether CBF, as assessed by response of middle cerebral arteryblood flow velocity (BFV) is altered in end stage liver disease (ESLD). METHODS: BFV was measured by transcranial Doppler which permitted on line, realtime evaluation of mean BFV (45+17 cardiac cycles/pt/state). 11ESLD patients and 10 normal centrals (CNT) were studied. BFV was measured sequentially at baseline (BL), 100% 02 (HiO2), hypocapnic (HV) and hypereapnic 0"IC) states. Endtidal CO2 (EtCO2), SaO2, mean arterial pressure (MAP) and EKG (HR) were continuously monitored. Subjects were studied somi-recumbeut~ breathing through a mouthpiece (nosoclip in place) and semi-closed anesthesia circuit. Data were analyzed using 2-way ANOVA and differences between CNT and ESLD reported significant at p<.05. RESULTS: Across all slates, SaO2 was the same, HR higher, and MAP lower in ESLD. EtcCn %A vs BL MCA %,% vs Data= (mmHg) EToO2 BFV BLBFV Mean+SD 47.2+4.0 BL CNT 35.2+0.9 39.8+4.4* ESLD 30.0+_1.6* HiO2 CNT 35.4+_0.9 0.7+1.9 47.5+_4.0 1.0+3.1 ESLD 29+_t.6" -3.4+_1.5 37.2+_4.2* -6.6+_2.5 22.7+0.9 -35.4+2.0 30.4+_3.3 -36.3+3.9 HV CNT ESLD 19.4+_1.2" -35.2+1.9 27.3+3.4* -32.8+_3.3 46.6+_0.6 33.0+_3.6 67.3+4.5 45.2+_6.2 HC CNT ESLD 40.6+_2.1" 35.6+_1.4 54.6+_6.8* 34.2+_5.7 BFV and EtCO2 were lower in ESLD in all states. The percent change in BFV with change in EtCO2 was similar in ESLD and CNT groups. CONCLUSION: Our findings clearly demonstrate preservation of normal BFV in response to alterations in the EtCO2. The baseline decrease in BFV in ESLD may be due to cerebral vasoconstriction possibly related to hyperventilation` intrapulmonary shunts, or systemic peripheral vasedilatation.
615
ABSENCE OF UNIQUE HBV MUTATION IN N. AMERICAN PATIENTS WITH FULMINANT HEPATITIS B. H Bozkava. R Reddv*. El lones. B Avola. L leffers*. D Bernstein*. M De Medina*. E Schiff*. ASF Lok. T u l a n e Unlv a n d VA Med Ctr, New Orleans, LA., Univ o f Miami*, Miami, FL Pre-core s t o p c o d o n m u t a t i o n (A1896) a n d m u t a t i o n s involving t h e 2 n d T-A r i c h r e g i o n (T1762 & A 1 7 6 4 ) o f the core p r o m o t e r h a v e b e e n r e p o r t e d to b e m o r e f r e q u e n t l y f o u n d ha J a p a n e s e a n d E u r o p e a n p a t i e n t s (pt) with f u l m i n a n t hepatitis B (FH). We p r e v i o u s l y r e p o r t e d t h a t A 1 8 9 6 is o n l y f o u n d in pt with T a n d n o t t h o s e with C at n t 1 8 5 8 d u e to b a s e - p a i r i n g in the p r e g e n o m e e n c a p s i d a t i o n signal (e) . Aims : To c o m p a r e the p r e v a l e n c e a n d n a t u r e of the s e q u e n c e v a r i a t i o n s in t h e HBV p r e - c o r e J c o r e a n d core p r o m o t e r r e g i o n in A m e r i c a n p t with FH vs t h o s e with a c u t e self-limiting h e p a t i t i s B (AID. Methods : Sera w e r e a n a l y z e d b y d i r e c t s e q u e n c i n g o f c o r e p r o m o t e r , p r e core, a n d e n t i r e core r e g i o n s of PCR a m p l i f i e d HBV DNA. Results : C o r e - o r o m o t e r - 5 FH a n d 11 All p t w e r e s t u d i e d . 3 FH a n d 6 AH p t h a d a d d i t i o n a l s m a l l e r b a n d ( s ) p o s s i b l y d u e to deletions. No significant c h a n g e was d e t e c t e d in t h e 1st & 3 r d T-A r i c h regions. C h a n g e s in the 2 n d T-A r i c h r e g i o n i n c l u d e : coo c c u r r e n c e o f T 1 7 6 2 & A 1 7 6 4 in 1 FH a n d 2 AH pt, a n d AT / C / G 1 7 6 0 in 1 FH a n d 3 AH pt. P r e - c o r e - 11 FH a n d 15 AH pt w e r e studied. 3 / 4 FH a n d 0 / 2 All pt w h o h a d T at n t 1858 h a d A1896. Of the 2 0 pt w i t h C at n t 1858, n o n e h a d A1896; 2 AH p t h a d co-existent G-T 1 8 6 2 & G-A 1 8 8 8 involving the b u l g e a n d u p p e r s t e m of ¢; a n o t h e r AH p t h a d G-A 1 8 8 8 & G-A 1 8 9 1 . Core # e n e : 8 FH a n d 15 AH pt w e r e studied. All 8 FH p t b u t o n l y 9 (60%) AH pt h a d > 1 a m i n o a c i d (aa) s u b s t i t u t i o n . T h e m e a n no. o f a a c h a n g e s in p t with FH vs AH w e r e 3.9+2.9 vs 1.5+1.8 (p=O.02). Conclusions : Pre-core s t o p c o d o n m u t a t i o n (A1896) was r a r e l y f o u n d in A m e r i c a n p t with FH d u e to t h e p r e d o m i n a n c e o f C at nt 1858. A p a r t f r o m a significantly h i g h e r r a t e of a a s u b s t i t u t i o n s in t h e c o r e gene, t h e r e was n o d i f f e r e n c e in p r e v a l e n c e o r n a t u r e of n t o r a a c h a n g e s in t h e HBV core p r o m o t e r , pre-core, a n d core r e g i o n s in p t with FH vs t h o s e with AH.
HEPATOLOGYOctober 1995
616
H E P A T I T I S B VIRUS CORE P R O M O T E R S E Q U E N C E A N A L Y S I S IN FULMINANT A N D CHRONIC H E P A T I T I S B T Laskus. MJ Nowicki*. DH Persinqt, J R a k e l a U n i v e r s i t y of P i t t s b u r g h Medical Center, Pittsburgh; * U n i v e r s i t y of Southern C a l i f o r n i a School of M e d i c i n e and t Mayo Clinic and Foundation, R o c h e s t e r N o n s e n s e m u t a t i o n s w i t h i n the p r e c o r e r e g i o n of hepatitis B virus (HBV) are r a r e l y e n c o u n t e r e d in fulminanthepatitis B p a t i e n t s in the USA. It has r e c e n t l y been r e p o r t e d that two point m u t a t i o n s w i t h i n the hepatitis B virus (HBV) core p r o m o t e r r e g i o n (A to T at p o s i t i o n 1762 and G to A at p o s i t i o n 1764) are a s s o c i a t e d with fulminant h e p a t i t i s and lead to H B e A g n e g a t i v e phenotype. Methods: Core p r o m o t e r region of HBV was a m p l i f i e d by PCR and d i r e c t l y s e q u e n c e d in 94 patients; 37 had fulminant hepatitis, 20 h a d acute self-limited hepatitis, 30 had chronic h e p a t i t i s and 7 p a t i e n t s h a d end-stage cirrhosis. Results: Core p r o m o t e r region was found to be h e t e r o g e n o u s and no specific changes c o r r e l a t e d w i t h H B e / a n t i - H B e status or survival in fulminant h e p a t i t i s patients. Substitutions at p o s i t i o n s 1762 and 1764 were found in HBV strains from four fulminant h e p a t i t i s p a t i e n t s (10%), from two p a t i e n t s w i t h selflimited h e p a t i t i s (10%), from eight (27%) chronic h e p a t i t i s patients and in 5 out of 7 e n d - s t a g e cirrhosis patients. The m a j o r i t y of these p a t i e n t s were HBeAg positive. Conclusions: M u t a t i o n s at p o s i t i o n s 1762 and 1764 are r a r e l y s e e n in HBV strains from f u l m i n a n t h e p a t i t i s B patients in the USA but are c o m m o n in chronic hepatitis. Even when present, they a p p e a r to be insufficient to lead to H B e A g n e g a t i v e phenotype.
613
AASLD ABSTRACTS
PRESERVATION OF CO2-DEPENDENT REGULATION OF CEREBRAL ARTERIAL BLOOD FLOW VELOCITY IN END STAGE LIVER DISEASE. JJ Katz. MS Mandell, B Barton, RM House. BM Bilir. GT Eversan. S Zamudin. Depts. of Anesth., Psychiatry, Medicine, University of Colorado School of Medicine, Denver.
614 HBV CORE PROMOTOR MUTATIONS CAUSE ENHANCED VIRAL REPLICATION IN ASSOCIATION WITH FULMINANT HEPATITIS. TF Baumert. SA Roonrs. K Haseaewa. and TJ Liarm. GI Unit, Dept of Medicine, Mass General Hosp and Harvard Medical School, Boston, MA. Mutations in hepatitis B virus (HBV) have been associated with the development of tulminant hepatitis. However, very few studies have addressed the functional relevance of these mutations. We recently cloned, sequenced, and characterized functionally the viral genome of a HBV strain associated with an epidemic of fulminant hepatitis (FH strain). Besides the well described precore mutations at nucleotide (nt) positions 1898 and 1901, numerous other mutations in various regions of the HBV genome were found. This HBV strain was functionally characterized by an increased encapsidation of pregenomic RNA leading to highly enhanced replication, which was not a result of the above mentioned precore mutations. We mapped the mutation(s) responsible for the enhanced replication of the FH strain to the core promotor. In order to define the responsible mutations, we introduced various mutations of the FH strain into a wild-type construct by site-directed mutagenesis. Viral antigen synthesis, transcription, encapsidation of pregenomic viral RNA, and replication of these mutant constructs were analyzed in comparison with those of wild-type strains. Analysis of viral replication showed that only the combined mutations of C to T at nt 1768 and T to A at nt 1770 directed the production of high level of coreassociated replicative intermediates. Similar to the FH strain, this mutant also displayed an enhanced encapsidation of pregenomic RNA. Cells transfected with wild-type and mutant HBV DNAs showed similar levels of total viral RNA and viral proteins, indicating that transcription and protein expression is not affected significantly by the introduced mutations; To extend our findings, we sequenced the core promotor region of six other viral strains from patients with fulminant hepatitis. Five out of six viral strains contained either the above mentioned or other mutations in the core promotor. Conclusion: Our studies show that two adjacent mutations in the core promolor lead to enhanced viral replication on the basis of increased encapsidation. Furthermore, functional characterization of the core promotor mutant suggests that mutations in this genomic region of HBV may play an important role in the pathogenesis of fulminant hepatic failure.
Although portal shunting and humoral mediators are implicated in portal-systemic encephalopathy, other factors, such as impaired astoregulation of cerebral blood flow (CBF) may contribute. We asked whether CBF, as assessed by response of middle cerebral arteryblood flow velocity (BFV) is altered in end stage liver disease (ESLD). METHODS: BFV was measured by transcranial Doppler which permitted on line, realtime evaluation of mean BFV (45+17 cardiac cycles/pt/state). 11ESLD patients and 10 normal centrals (CNT) were studied. BFV was measured sequentially at baseline (BL), 100% 02 (HiO2), hypocapnic (HV) and hypereapnic 0"IC) states. Endtidal CO2 (EtCO2), SaO2, mean arterial pressure (MAP) and EKG (HR) were continuously monitored. Subjects were studied somi-recumbeut~ breathing through a mouthpiece (nosoclip in place) and semi-closed anesthesia circuit. Data were analyzed using 2-way ANOVA and differences between CNT and ESLD reported significant at p<.05. RESULTS: Across all slates, SaO2 was the same, HR higher, and MAP lower in ESLD. EtcCn %A vs BL MCA %,% vs Data= (mmHg) EToO2 BFV BLBFV Mean+SD 47.2+4.0 BL CNT 35.2+0.9 39.8+4.4* ESLD 30.0+_1.6* HiO2 CNT 35.4+_0.9 0.7+1.9 47.5+_4.0 1.0+3.1 ESLD 29+_t.6" -3.4+_1.5 37.2+_4.2* -6.6+_2.5 22.7+0.9 -35.4+2.0 30.4+_3.3 -36.3+3.9 HV CNT ESLD 19.4+_1.2" -35.2+1.9 27.3+3.4* -32.8+_3.3 46.6+_0.6 33.0+_3.6 67.3+4.5 45.2+_6.2 HC CNT ESLD 40.6+_2.1" 35.6+_1.4 54.6+_6.8* 34.2+_5.7 BFV and EtCO2 were lower in ESLD in all states. The percent change in BFV with change in EtCO2 was similar in ESLD and CNT groups. CONCLUSION: Our findings clearly demonstrate preservation of normal BFV in response to alterations in the EtCO2. The baseline decrease in BFV in ESLD may be due to cerebral vasoconstriction possibly related to hyperventilation` intrapulmonary shunts, or systemic peripheral vasedilatation.
615
ABSENCE OF UNIQUE HBV MUTATION IN N. AMERICAN PATIENTS WITH FULMINANT HEPATITIS B. H Bozkava. R Reddv*. El lones. B Avola. L leffers*. D Bernstein*. M De Medina*. E Schiff*. ASF Lok. T u l a n e Unlv a n d VA Med Ctr, New Orleans, LA., Univ o f Miami*, Miami, FL Pre-core s t o p c o d o n m u t a t i o n (A1896) a n d m u t a t i o n s involving t h e 2 n d T-A r i c h r e g i o n (T1762 & A 1 7 6 4 ) o f the core p r o m o t e r h a v e b e e n r e p o r t e d to b e m o r e f r e q u e n t l y f o u n d ha J a p a n e s e a n d E u r o p e a n p a t i e n t s (pt) with f u l m i n a n t hepatitis B (FH). We p r e v i o u s l y r e p o r t e d t h a t A 1 8 9 6 is o n l y f o u n d in pt with T a n d n o t t h o s e with C at n t 1 8 5 8 d u e to b a s e - p a i r i n g in the p r e g e n o m e e n c a p s i d a t i o n signal (e) . Aims : To c o m p a r e the p r e v a l e n c e a n d n a t u r e of the s e q u e n c e v a r i a t i o n s in t h e HBV p r e - c o r e J c o r e a n d core p r o m o t e r r e g i o n in A m e r i c a n p t with FH vs t h o s e with a c u t e self-limiting h e p a t i t i s B (AID. Methods : Sera w e r e a n a l y z e d b y d i r e c t s e q u e n c i n g o f c o r e p r o m o t e r , p r e core, a n d e n t i r e core r e g i o n s of PCR a m p l i f i e d HBV DNA. Results : C o r e - o r o m o t e r - 5 FH a n d 11 All p t w e r e s t u d i e d . 3 FH a n d 6 AH p t h a d a d d i t i o n a l s m a l l e r b a n d ( s ) p o s s i b l y d u e to deletions. No significant c h a n g e was d e t e c t e d in t h e 1st & 3 r d T-A r i c h regions. C h a n g e s in the 2 n d T-A r i c h r e g i o n i n c l u d e : coo c c u r r e n c e o f T 1 7 6 2 & A 1 7 6 4 in 1 FH a n d 2 AH pt, a n d AT / C / G 1 7 6 0 in 1 FH a n d 3 AH pt. P r e - c o r e - 11 FH a n d 15 AH pt w e r e studied. 3 / 4 FH a n d 0 / 2 All pt w h o h a d T at n t 1858 h a d A1896. Of the 2 0 pt w i t h C at n t 1858, n o n e h a d A1896; 2 AH p t h a d co-existent G-T 1 8 6 2 & G-A 1 8 8 8 involving the b u l g e a n d u p p e r s t e m of ¢; a n o t h e r AH p t h a d G-A 1 8 8 8 & G-A 1 8 9 1 . Core # e n e : 8 FH a n d 15 AH pt w e r e studied. All 8 FH p t b u t o n l y 9 (60%) AH pt h a d > 1 a m i n o a c i d (aa) s u b s t i t u t i o n . T h e m e a n no. o f a a c h a n g e s in p t with FH vs AH w e r e 3.9+2.9 vs 1.5+1.8 (p=O.02). Conclusions : Pre-core s t o p c o d o n m u t a t i o n (A1896) was r a r e l y f o u n d in A m e r i c a n p t with FH d u e to t h e p r e d o m i n a n c e o f C at nt 1858. A p a r t f r o m a significantly h i g h e r r a t e of a a s u b s t i t u t i o n s in t h e c o r e gene, t h e r e was n o d i f f e r e n c e in p r e v a l e n c e o r n a t u r e of n t o r a a c h a n g e s in t h e HBV core p r o m o t e r , pre-core, a n d core r e g i o n s in p t with FH vs t h o s e with AH.
HEPATOLOGYOctober 1995
616
H E P A T I T I S B VIRUS CORE P R O M O T E R S E Q U E N C E A N A L Y S I S IN FULMINANT A N D CHRONIC H E P A T I T I S B T Laskus. MJ Nowicki*. DH Persinqt, J R a k e l a U n i v e r s i t y of P i t t s b u r g h Medical Center, Pittsburgh; * U n i v e r s i t y of Southern C a l i f o r n i a School of M e d i c i n e and t Mayo Clinic and Foundation, R o c h e s t e r N o n s e n s e m u t a t i o n s w i t h i n the p r e c o r e r e g i o n of hepatitis B virus (HBV) are r a r e l y e n c o u n t e r e d in fulminanthepatitis B p a t i e n t s in the USA. It has r e c e n t l y been r e p o r t e d that two point m u t a t i o n s w i t h i n the hepatitis B virus (HBV) core p r o m o t e r r e g i o n (A to T at p o s i t i o n 1762 and G to A at p o s i t i o n 1764) are a s s o c i a t e d with fulminant h e p a t i t i s and lead to H B e A g n e g a t i v e phenotype. Methods: Core p r o m o t e r region of HBV was a m p l i f i e d by PCR and d i r e c t l y s e q u e n c e d in 94 patients; 37 had fulminant hepatitis, 20 h a d acute self-limited hepatitis, 30 had chronic h e p a t i t i s and 7 p a t i e n t s h a d end-stage cirrhosis. Results: Core p r o m o t e r region was found to be h e t e r o g e n o u s and no specific changes c o r r e l a t e d w i t h H B e / a n t i - H B e status or survival in fulminant h e p a t i t i s patients. Substitutions at p o s i t i o n s 1762 and 1764 were found in HBV strains from four fulminant h e p a t i t i s p a t i e n t s (10%), from two p a t i e n t s w i t h selflimited h e p a t i t i s (10%), from eight (27%) chronic h e p a t i t i s patients and in 5 out of 7 e n d - s t a g e cirrhosis patients. The m a j o r i t y of these p a t i e n t s were HBeAg positive. Conclusions: M u t a t i o n s at p o s i t i o n s 1762 and 1764 are r a r e l y s e e n in HBV strains from f u l m i n a n t h e p a t i t i s B patients in the USA but are c o m m o n in chronic hepatitis. Even when present, they a p p e a r to be insufficient to lead to H B e A g n e g a t i v e phenotype.