HBx M130K and V131I (T-A) mutations in HBV genotype F during a follow-up study in chronic carriers

Abstract

Background: Around 400 million people worldwide are chronically infected with Hepatitis B virus (HBV). An estimated 10% of these chronic patients develop progressive liver damage including cirrhosis and Hepatocellular Carcinoma (HCC). The HBx gene encodes a protein of 154 amino acids which is a transactivator and has been associated with HBV pathogenesis. A change in the amino acid sequences at positions 130 and 131 in the HBV-X protein (M130K and V131I) produced by T-A point mutations at the nucleic acids level has been associated with severe liver damage and HCC in patients from China and Africa. Further, such changes have been proposed as a prognostic marker for progressive liver damage and HCC. The purpose of this study was to determine if T-A mutations are present in HBV chronic carriers with genotype F (the major genotype in Costa Rica) and further, if these mutations are associated with HBV disease progression in Costa Rica HBV patients from 1972 to 1985.

Results: Serum samples from 50 HBV positive individuals were amplified and directly sequenced, 48 belonged to genotype F, 1 from genotype D and another was classified as D or E. T-A mutations were absent in 17 acute patients who recovered, but was present in 12 of 29 chronic carrier samples (42.8%), in one sample the T-A mutations were detected as early as 29 days after clinical onset of disease. In 17 carriers with available liver biopsies, T-A mutations were found in 8 sera of 13 (61.5%) classified as moderate or severe, and none in 4 biopsies with mild liver damage. However, it was not possible to demonstrate a statistical association between the presence of T-A mutations and moderate/severe liver damage, using a Fischer exact test, 1 tail, p = 0.05. In 4 patients HCC was diagnosed, and 2 of them presented the T-A mutations in their sera.

Conclusion: T-A mutations were found in HBV genotype F in chronic carriers but not in patients who recovered from acute infection. These mutations could be developing early during infection although the possibility of infection with the mutant virus could not be excluded. More studies are necessary to establish if the T-A mutation can be used as a prognostic marker for severity of liver disease in patients infected with HBV.

Figure 1

(A) – Persistent chronic hepatitis, Knodell index ≤ 2. Photomicrograph of liver showing chronic hepatitis with minimal activity. Hepatocytes showing regenerative features are seen, with minimal inflammation and scattered ground- glass hepatocytes. Cobblestone arrangement (diffuse regeneration) with Hadziyannis cells and without necrosis or fibrosis. (H&E 250×). (B) – Mild lobular chronic hepatitis, Knodell index 3–4. Photomicrograph of liver showing chronic hepatitis with mild activity. Spotty hepatocyte necrosis is seen in a lobular pattern with focal lymphocytic infiltration. Lesions are characterized by focal necrosis, conserved sinusoidal and trabecular patterns, lobular, portal, and focal lymphocytic infiltrated. (H&E 400×). (C) – Moderate lobular chronic hepatitis, Knodell index > 4. Photomicrograph of liver showing chronic hepatitis with moderate activity. There is portal chronic inflammation, focal interface hepatitis and periportal fibrous septa. Portal chronic swollen periportal apoptosis, post-necrosis fibrous interportal bridges. Nodular regeneration (pre-cirrhosis). (H&E 250×)

Figure 2

Sample deletions treated with Ssp I restriction enzymes. Recognition site of the enzyme SspI in the sequences with 8 bp deletion (left). In the right, samples with presumed deletions were run in a 3% agarose gel. Each pair of lines have the same sample treated with and without the Ssp I enzyme. An HIV sample having the AATATT site was used as positive control in lanes 1 and 2, sample 1430 (616 bp) lanes 3 and 4 (negative control), 1000 bp ladder marker lane 5, sample 6290 lanes 6 and 7, sample 467 lanes 8 and 9 sample 6516 lines 10 and 11, sample 6541 lanes 12 and 13. The samples 467 and 6516 treated with SspI presented two bands of 507 and 109 bp, lanes 8 and 10 (arrows) confirming the deletion. Details in sequence are: