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Association of the core clustering mutations (codon 21-34) and the severity of chronic hepatitis B in Korean patients.

Koh KC, Lee HS, Kim CY - Korean J. Intern. Med. (1995)

Bottom Line: A total of 181 nucleotide substitutions were found in the HBV core gene from the 15 CH-B patients, of which 23 were missense and 158 were silent.Two mutational hot spots (MHS), codons 21-34 (MHS1) and codons 85-100 (MHS2), were found in the deduced amino acid alignment of the core gene.In comparison, of 7 CAH patients, 3 showed them both in MHS1 and MHS2, 1 only in MHS1, and 1 only in MHS2; thus, missense mutation in MHS1 was exclusively found in patient with CAH.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Seoul National University College of Medicine, Korea.

ABSTRACT

Objectives: There are regions in the core gene of hepatitis B virus (HBV) where missense mutations are clustered, and mutations in that region are related to severe liver disease. However, there were some differences of the major regions for mutation clustering among ethnic groups. To explore the phenomenon of clustering mutations in Korean patients with chronic HBV infection and to elucidate the correlation between clustering mutation region of the core gene and the severity of liver damage, we analyzed the precore/core gene sequence of HBV in the sera from fifteen chronic hepatitis B (CH-B) patients.

Methods: We analysed the HBV precore and core sequences in the sera obtained from fifteen patients (14 males and 1 female, mean age 30.0 years) with biopsy-proven CH-B. The patients were divided into two groups according to the pathological severity of CH-B; namely, group I consisted of 8 patients with chronic persistent hepatitis (CPH), and group II included 7 patients with chronic active hepatitis (CAH). After extraction of HBV DNA from each serum by proteinase K and phenol-chloroform solution, the entire precore and core region of HBV was amplified by PCR, and then the PCR products were subjected to direct sequencing using thermostable DNA polymerase. Fisher's exact test and Mann-Whitney U test were used for statistical analysis.

Results: A total of 181 nucleotide substitutions were found in the HBV core gene from the 15 CH-B patients, of which 23 were missense and 158 were silent. The nucleotide and amino acid substitution rates were not significantly different between the two groups (p > 0.05). Two mutational hot spots (MHS), codons 21-34 (MHS1) and codons 85-100 (MHS2), were found in the deduced amino acid alignment of the core gene. The alteration rate of amino acid residue in these regions were 2.857 x 10(-2) and 5.000 x 10(-2), respectively. Of 8 CPH patients, 5 showed missense mutations only in MHS2. In comparison, of 7 CAH patients, 3 showed them both in MHS1 and MHS2, 1 only in MHS1, and 1 only in MHS2; thus, missense mutation in MHS1 was exclusively found in patient with CAH.

Conclusions: There were two mutation clusterings in the core region of adr subtype of HBV from Korean CH-B patients. Mutations in MHS1 (codon 21-34), but not in MHS2 (codon 85-100), are more likely to be related to the severity of CH-B. A longitudinal study using sequential samples is warranted to further clarify the role of MHS1 in the pathogenesis of more severe CH-B.

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Related in: MedlinePlus

The nonstandard gel-loading format. Using the nonstandard gel-loding format, all sequences generated from a single primer were run side-by-side (GGGAAATTTCCC). The mutations were detected based on differences in the banding patterns. The arrowheads in the figure denote mutations. The standard gel-loading format is displayed on the left side for comparison.
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f1-kjim-10-2-87-2: The nonstandard gel-loading format. Using the nonstandard gel-loding format, all sequences generated from a single primer were run side-by-side (GGGAAATTTCCC). The mutations were detected based on differences in the banding patterns. The arrowheads in the figure denote mutations. The standard gel-loading format is displayed on the left side for comparison.

Mentions: We prepared several sequencing primers to sequence bi-directionally: sense primers, P1 (nt 1741–1758, 5′-GGGAGGAGATTAGGTTAA-3′), P2 (nt 1843–1867, 5′-CATGTTCATGTCCTACTGTTCA-3′), P3 (nt 2047–2071, 5′-CTCATCATACAGCACTCAGGCAAGC-3′), P4 (nt 2292–2310, 5′-TACAGACCACCAAATGCCC-3′); antisense primers, M1 (nt 2472–2497, 5′-AGAATAAAGCCCAGTAAAGTTTCCC-3′), M2 (nt 2287–2310, 5′-GGGCATTTGGTGGTCTGTA-3′), M3 (nt 2114–2138, 5′-TGCTGGGTCTTCCAAATTACTTCCC-3′), M4 (nt 2047–2067, 5′-GCCTGAGTGCT GTATGATGAG-3′), M5 (nt 1910–1934, 5′-AGAAGCTCCAAATTCTTTATACG-3′). Each sequencing primer was radiolabeled with γ-32P-ATP using T4 polynucleotide kinase. From 40 to 100 fmol of purified PCR product and 1.5 pmol of each radiolabeled primer was used for cycle sequencing reaction19) using thermostable DNA polymerase (fmol™ sequencing system, Promega, Madison, WI, USA). The reaction products were analyzed on a 8% sequencing gel. For ease of detection of mutations, we used a nontraditional gel-loading format20) in which all G-terminated reaction products from a single primer are loaded side-by-side, followed by all the A-terminated reactions, etc. (GGGGAAAATTTTCCCC) (Fig. 1). This format allowed mutations to be detected by virtue of the differences in the banding patterns, rather than by reading each sequence and comparing it to the known sequence. HBsAg subtypes in the fifteen patients were all adr. When four previously reported amino acid residues of the HBV with adr subtype17,21–23) were aligned, the sequences showed well conserved amino acid residues of core peptides and the only difference was in the codon 169. Since, in all patients, the deduced amino acid residue in the codon 169 was consistent with all 3 sequences reported in Japan, we used that sequence as a prototype HBV.


Association of the core clustering mutations (codon 21-34) and the severity of chronic hepatitis B in Korean patients.

Koh KC, Lee HS, Kim CY - Korean J. Intern. Med. (1995)

The nonstandard gel-loading format. Using the nonstandard gel-loding format, all sequences generated from a single primer were run side-by-side (GGGAAATTTCCC). The mutations were detected based on differences in the banding patterns. The arrowheads in the figure denote mutations. The standard gel-loading format is displayed on the left side for comparison.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4532043&req=5

f1-kjim-10-2-87-2: The nonstandard gel-loading format. Using the nonstandard gel-loding format, all sequences generated from a single primer were run side-by-side (GGGAAATTTCCC). The mutations were detected based on differences in the banding patterns. The arrowheads in the figure denote mutations. The standard gel-loading format is displayed on the left side for comparison.
Mentions: We prepared several sequencing primers to sequence bi-directionally: sense primers, P1 (nt 1741–1758, 5′-GGGAGGAGATTAGGTTAA-3′), P2 (nt 1843–1867, 5′-CATGTTCATGTCCTACTGTTCA-3′), P3 (nt 2047–2071, 5′-CTCATCATACAGCACTCAGGCAAGC-3′), P4 (nt 2292–2310, 5′-TACAGACCACCAAATGCCC-3′); antisense primers, M1 (nt 2472–2497, 5′-AGAATAAAGCCCAGTAAAGTTTCCC-3′), M2 (nt 2287–2310, 5′-GGGCATTTGGTGGTCTGTA-3′), M3 (nt 2114–2138, 5′-TGCTGGGTCTTCCAAATTACTTCCC-3′), M4 (nt 2047–2067, 5′-GCCTGAGTGCT GTATGATGAG-3′), M5 (nt 1910–1934, 5′-AGAAGCTCCAAATTCTTTATACG-3′). Each sequencing primer was radiolabeled with γ-32P-ATP using T4 polynucleotide kinase. From 40 to 100 fmol of purified PCR product and 1.5 pmol of each radiolabeled primer was used for cycle sequencing reaction19) using thermostable DNA polymerase (fmol™ sequencing system, Promega, Madison, WI, USA). The reaction products were analyzed on a 8% sequencing gel. For ease of detection of mutations, we used a nontraditional gel-loading format20) in which all G-terminated reaction products from a single primer are loaded side-by-side, followed by all the A-terminated reactions, etc. (GGGGAAAATTTTCCCC) (Fig. 1). This format allowed mutations to be detected by virtue of the differences in the banding patterns, rather than by reading each sequence and comparing it to the known sequence. HBsAg subtypes in the fifteen patients were all adr. When four previously reported amino acid residues of the HBV with adr subtype17,21–23) were aligned, the sequences showed well conserved amino acid residues of core peptides and the only difference was in the codon 169. Since, in all patients, the deduced amino acid residue in the codon 169 was consistent with all 3 sequences reported in Japan, we used that sequence as a prototype HBV.

Bottom Line: A total of 181 nucleotide substitutions were found in the HBV core gene from the 15 CH-B patients, of which 23 were missense and 158 were silent.Two mutational hot spots (MHS), codons 21-34 (MHS1) and codons 85-100 (MHS2), were found in the deduced amino acid alignment of the core gene.In comparison, of 7 CAH patients, 3 showed them both in MHS1 and MHS2, 1 only in MHS1, and 1 only in MHS2; thus, missense mutation in MHS1 was exclusively found in patient with CAH.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Seoul National University College of Medicine, Korea.

ABSTRACT

Objectives: There are regions in the core gene of hepatitis B virus (HBV) where missense mutations are clustered, and mutations in that region are related to severe liver disease. However, there were some differences of the major regions for mutation clustering among ethnic groups. To explore the phenomenon of clustering mutations in Korean patients with chronic HBV infection and to elucidate the correlation between clustering mutation region of the core gene and the severity of liver damage, we analyzed the precore/core gene sequence of HBV in the sera from fifteen chronic hepatitis B (CH-B) patients.

Methods: We analysed the HBV precore and core sequences in the sera obtained from fifteen patients (14 males and 1 female, mean age 30.0 years) with biopsy-proven CH-B. The patients were divided into two groups according to the pathological severity of CH-B; namely, group I consisted of 8 patients with chronic persistent hepatitis (CPH), and group II included 7 patients with chronic active hepatitis (CAH). After extraction of HBV DNA from each serum by proteinase K and phenol-chloroform solution, the entire precore and core region of HBV was amplified by PCR, and then the PCR products were subjected to direct sequencing using thermostable DNA polymerase. Fisher's exact test and Mann-Whitney U test were used for statistical analysis.

Results: A total of 181 nucleotide substitutions were found in the HBV core gene from the 15 CH-B patients, of which 23 were missense and 158 were silent. The nucleotide and amino acid substitution rates were not significantly different between the two groups (p > 0.05). Two mutational hot spots (MHS), codons 21-34 (MHS1) and codons 85-100 (MHS2), were found in the deduced amino acid alignment of the core gene. The alteration rate of amino acid residue in these regions were 2.857 x 10(-2) and 5.000 x 10(-2), respectively. Of 8 CPH patients, 5 showed missense mutations only in MHS2. In comparison, of 7 CAH patients, 3 showed them both in MHS1 and MHS2, 1 only in MHS1, and 1 only in MHS2; thus, missense mutation in MHS1 was exclusively found in patient with CAH.

Conclusions: There were two mutation clusterings in the core region of adr subtype of HBV from Korean CH-B patients. Mutations in MHS1 (codon 21-34), but not in MHS2 (codon 85-100), are more likely to be related to the severity of CH-B. A longitudinal study using sequential samples is warranted to further clarify the role of MHS1 in the pathogenesis of more severe CH-B.

Show MeSH
Related in: MedlinePlus