Quadruple mutation GCAC1809-1812TTCT acts as a biomarker in healthy European HBV carriers
Kai-Henrik Peiffer, Catrina Spengler, Michael Basic, Bingfu Jiang, Lisa Kuhnhenn, Wiebke Obermann, Tobias Zahn, Mirco Glitscher, Alessandro Loglio, Floriana Facchetti, Gert Carra, Alica Kubesch, Johannes Vermehren, Viola Knop, Christiana Graf, Julia Dietz, Fabian Finkelmeier, Eva Herrmann, Jonel Trebicka, Arnold Grünweller, Stefan Zeuzem, Christoph Sarrazin, Pietro Lampertico, Eberhard Hildt
Kai-Henrik Peiffer, Catrina Spengler, Michael Basic, Bingfu Jiang, Lisa Kuhnhenn, Wiebke Obermann, Tobias Zahn, Mirco Glitscher, Alessandro Loglio, Floriana Facchetti, Gert Carra, Alica Kubesch, Johannes Vermehren, Viola Knop, Christiana Graf, Julia Dietz, Fabian Finkelmeier, Eva Herrmann, Jonel Trebicka, Arnold Grünweller, Stefan Zeuzem, Christoph Sarrazin, Pietro Lampertico, Eberhard Hildt
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Research Article Hepatology Virology

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.

Authors

Kai-Henrik Peiffer, Catrina Spengler, Michael Basic, Bingfu Jiang, Lisa Kuhnhenn, Wiebke Obermann, Tobias Zahn, Mirco Glitscher, Alessandro Loglio, Floriana Facchetti, Gert Carra, Alica Kubesch, Johannes Vermehren, Viola Knop, Christiana Graf, Julia Dietz, Fabian Finkelmeier, Eva Herrmann, Jonel Trebicka, Arnold Grünweller, Stefan Zeuzem, Christoph Sarrazin, Pietro Lampertico, Eberhard Hildt

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Figure 2

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

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Diminished synthesis of HBsAg by GCAC1809-1812TTCT in vitro. (A) Overvie...
(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.