Evolutionary Changes of Hepatitis B Virus Pre-S Mutations Prior to Development of Hepatocellular Carcinoma

Abstract

Background and aims: Deletions/mutations in the hepatitis B virus (HBV) pre-S region have been associated with hepatocellular carcinoma (HCC). We aimed to study the evolutionary changes of pre-S mutations prior to HCC development.

Methods: We studied the HBV pre-S sequences at 1 to 10 years preceding diagnosis of HCC in 74 patients with HBV-related HCC (HCC group). 148 chronic hepatitis B patients matched for sex and age in 2:1 ratio, who had been followed up for at least 3 years without HCC (HCC-free group) were recruited as controls. 56 and 47 patients of HCC and HCC-free groups respectively had serially stored sera for longitudinally examination at 1-3 years, 4-6 years, 7-9 years and ≥10 years prior to the recruitment of the study.

Results: Compared to the HCC-free group, higher frequencies of pre-S deletions and point mutations (at 11 codons) were observed in the HCC group (p<0.05). Multiple logistic regression analysis showed that pre-S deletions, point mutations at codon 51 and 167 were independent factors associated with HCC. Longitudinal observation showed that pre-S deletions and most of the 11 HCC-associated pre-S point mutations existed at least 10 years before HCC development, and were more prevalent preceding HCC development in patients from HCC groups than HCC-free group. The number of HCC-associated pre-S point mutations increased over time preceding HCC development, and correlated positively with the time to HCC diagnosis (r = 0.220, p = 0.005).

Conclusions: High prevalence and cumulative evolution of pre-S mutations preceding HCC development suggested a possible carcinogenic role of pre-S mutations and their potential application in HCC risk prediction.

Fig 1. Mapping of pre-S mutations in HCC patients and HCC-free patients.

The HBV pre-S region contains immune epitopes and functional domains as depicted. The B-cell epitopes located at aa12-53, aa72-78, aa94-117, aa120-143 and aa157-167 of pre-S gene (overlap regions merged); The T-cell epitopes located at aa21-30, aa52-67, aa120-145 and aa163-172 of pre-S gene (overlap regions merged). The functional domains include the start codons of pre-S1 (aa1) and pre-S2 (aa120), hepatocyte binding site (Hb, aa21-47), S promoter (Sp, nt3045-3180), CCAAT box (nucleotide 3137–3141), topology domain (Topo) includes heat shock protein 70 binding site (aa74-118) and cytosolic anchorage determinant (aa81-105), nucleocapsid binding site (Nb, aa103-127), viral secretion domain (Vs, aa120-124), polymerized human serum albumin binding site (pHSA, aa122-135), transactivator domain (Trans, aa21-90, 120–172). The sequence of each sample is depicted as a block. The black blocks inside represent the deletions of amino acids. The patient identifier numbers are listed at the right.

Fig 2. The positive selection codons detected in genotype C sequences.

Prevalence of mutations at positive selected sites of genotype C sequences in HCC and HCC-free group.

Fig 3. Longitudinal observation of pre-S deletions over time prior to HCC development.

Each sequence is depicted as a block and its location relative to the timeline indicates the time points prior to diagnosis of HCC. Empty blocks represent sequences without pre-S deletions; whereas black blocks represent sequences with pre-S deletions. The patient identifier numbers are listed at the right.

Fig 4. Changes of HBV pre-S point mutations numbers prior to HCC development.

Bubble plot of cumulative number of HBV pre-S point mutations associated with HCC at 4 time points in HCC group (A) and HCC-free group (B). Time points 1–4 represent time points of more than ≥10 years, 7–9 years, 4–6 years and 1–3 years before HCC diagnosis, respectively. The size of the circles corresponds to the number of patients at each given time point detected with each given number of point mutation.