Quadruple mutation GCAC1809-1812TTCT acts as a biomarker in healthy European HBV carriers

Abstract

Many mutation analyses of the HBV genome have been performed in the search for new prognostic markers. However, the Kozak sequence preceding precore was covered only infrequently in these analyses. In this study, the HBV core promoter/precore region was sequenced in serum samples from European inactive HBV carriers. Quadruple mutation GCAC1809-1812TTCT was found with a high prevalence of 42% in the Kozak sequence preceding precore among all HBV genotypes. GCAC1809-1812TTCT was strongly associated with coexistence of basal core promoter (BCP) double mutation A1762T/G1764A and lower HBV DNA levels. In vitro GCAC1809-1812TTCT lead to drastically diminished synthesis of pregenomic RNA (pgRNA), precore mRNA, core, HBsAg, and HBeAg. Calculation of the pgRNA secondary structure suggests a destabilization of the pgRNA structure by A1762T/G1764A that was compensated by GCAC1809-1812TTCT. In 125 patients with HBV-related cirrhosis, GCAC1809-1812TTCT was not detected. While a strong association of GCAC1809-1812TTCT with inactive carrier status was observed, BCP double mutation was strongly correlated with cirrhosis, but this was only observed in absence of GCAC1809-1812TTCT. In conclusion, our data reveal that GCAC1809-1812TTCT is highly prevalent in inactive carriers and acts as a compensatory mutation for BCP double mutation. GCAC1809-1812TTCT seems to be a biomarker of good prognosis in HBV infection.

Keywords: Clinical practice; Hepatology; Molecular biology; Virology.

Figure 1. High prevalence of GCAC1809-1812TTCT and association with BCP double mutation A1762T/G1764A and lower HBV DNA levels in HBsAg carriers.

(A–C) Prevalence of (A) BCP double mutation A1762T/G1764A and mutations at nt1809–1812, (B) mutations at nt1809–1812 dependent on coexistence of A1762T/G1764A, and (C) mutations at nt1809–1812 dependent on coexistence of A1762T/G1764A among different GTs (GTA–GTE) in sera of 560 patients with a chronic HBV infection (HBsAg carriers) from the Albatros cohort. (D and E) Association of A1762T/G1764A and GCAC1809-1812TTCT with (D) HBV DNA levels and (E) qHBsAg levels in inactive carriers from the Albatros cohort. Data are shown as follows: median (line inside the box); first and third quartile (upper and lower limit of the box, respectively); and the highest and lowest values are represented by the top and bottom whiskers. A Kruskal-Willis test with a post hoc Dunn’s test were performed to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. BCP, basal core promoter; HBsAg, HBV surface antigen; GTs, genotypes.

Figure 2. Diminished synthesis of HBsAg by GCAC1809-1812TTCT in vitro.

(A) Overview of expressed genotype A genomes based on a 1.2-mer isolate from a patient of the Albatros cohort (TTCT3) with both the A1762T/G1764A (BCP) double mutation and the GCAC1809-1812TTCT (TTCT) quadruple mutation in core promoter and HBx (due to partially overlapping reading frame of HBV). 0 = absence of the mutation, 1 = mutation only in core promoter, 2 = mutation only in HBx, and 3 = mutation in both core promoter and HBx. With respect to the clinical data, all variants (TTCT1–3) contain the additional A1762T/G1764A BCP mutation. A construct without these 2 mutations (BCP0 plus TTCT0) was used as a reference. ^aFor analysis of extracellular DNA and HBeAg, an additional genome harboring GCAC1809-1812TTCT in HBx and core promoter but without the A1762T/G1764A BCP double mutation was used (BCP0/TTCT3). A 1.1-mer HBeAg WT genome and an HBeAg-negative genome harboring a G1896A/G1899A precore mutation were used as controls. (B and C) HBsAg-specific ELISA of (B) lysates, n = 4, and (C) supernatants, n = 3. Data are shown as follows: median (line inside the box), first and third quartile (upper and lower limit of the box, respectively) and the highest and lowest values are represented by the top and bottom whiskers. Multiple t test with the Holm-Šidák method was performed to correct for multiple group comparisons and to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. HBsAg, HBV surface antigen; BCP, basal core promoter.

Figure 3. Diminished synthesis of core protein and HBeAg by GCAC1809-1812TTCT in vitro.

(A) Western blot analysis using a core-specific antibody of lysates; as additional control, a different HBeAg-negative genotype A genome harboring precore double mutation G1896A/G1899A was used. (B and C) HBcAg- and HBeAg-specific ELISA of lysates and supernatants (n = 4 and n = 3, respectively). Data are shown as follows: median (line inside the box); first and third quartile (upper and lower limit of the box, respectively); and the highest and lowest values are represented by the top and bottom whiskers. Multiple t test with the Holm-Šidák method was performed to correct for multiple group comparisons and to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) CLSM analysis of transfected Huh 7 cells stained with the core-specific antibody MAB3120. HBsAg, HBV surface antigen.

Figure 4. Impaired viral replication by GCAC1809-1812TTCT in vitro.

(A) Northern blot analysis; as additional control, an HBeAg-negative genotype A genome harboring precore double mutation G1896A/G1899A was used. (B–D) Real-time PCR analyses of lysates for total 3.5 kb (B), pregenomic (C), or precore (D) transcripts. (E–G) DNA real-time PCR analyses of supernatants. (G) An additional an additional genome harboring GCAC1809-1812TTCT in HBx and core promoter but without the A1762T/G1764A BCP double mutation was used (BCP0/TTCT3). Values were normalized to BCP0/TTCT0 (B–E) and represent a total value of n = 3 (B–D), n = 7 (E and F), and n = 5 (G) independent experiments. In F, values of E were normalized to BCP3 and in G values were normalized to TTCT3. Data are shown as follows (B–G): median (line inside the box); first and third quartile (upper and lower limit of the box, respectively); and the highest and lowest values are represented by the top and bottom whiskers. Multiple t test with the Holm-Šidák method was performed to correct for multiple group comparisons and to determine statistical significance in B–E, a 2-tailed student t test was performed to determine statistical significance in F and G. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. HBsAg, HBV surface antigen; BCP, basal core promoter.

Figure 5. Thermodynamic destabilization of the pgRNA secondary structure by A1762T/G1764A and compensation by GCAC1809-1812TTCT.

(A–D) Calculation of the thermodynamic stability of nt1730–1930 pgRNA genomic region genome with the presence of (A) none of the mutations, (B) A1762T/G1764A, (C), A1762T/G1764A and GCAC1809-1812TTCT, and (D) GCAC1809-1812TTCT without A1762T/G1764A. The used HBV sequence was derived from an inactive carrier patient with the coexistence of A1762T/G1764A and GCAC1809-1812TTCT. Red bars indicate the positions in the sequences where the mutations are localized. BCP, basal core promoter; MFE, minimum free energy; GC, guanine–cytosine.

Figure 6. GCAC1809-1812TTCT is absent in patients with cirrhosis and strongly associated with inactive carrier status.

(A) Prevalence of BCP double mutation A1762T/G1764A and mutations at nt1809–1812 in patients with liver cirrhosis. (B and C) Association of A1762T/G1764A with or without GCAC1809-1812TTCT with (B) the presence of liver cirrhosis and (C) the presence of liver cirrhosis and HCC in comparison with inactive carriers. A χ² test was used for group comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. BCP, basal core promoter.