HBV preS deletion mapping using deep sequencing demonstrates a unique association with viral markers

Abstract

Aim: Deletions are observed frequently in the preS1/S2 region of hepatitis B virus (HBV) genome, in association with liver disease advancement. However, the most significant preS1/S2 region and its influences on viral markers are unclear.

Methods: The preS1/S2 HBV regions of 90 patients without antiviral therapy were subjected to deep sequencing and deleted regions influencing viral markers were investigated.

Results: From the deletion frequency analysis in each patient, deletions were observed most frequently in the preS2 codon 132-141 region. When the patients were divided into three groups (0-0.1%: n = 27, 0.1%-10%: n = 34, 10-100%: n = 29), based on the deletion frequency, FIB-4 (p < 0.01), HBV DNA (p < 0.01), HBcrAg (p < 0.01) and preS1/S2 start codon mutations (p < 0.01, both) were significantly associated with the deletion. When clinical and viral markers were investigated by multivariate analysis for their association with the deletion, FIB-4 (p < 0.05), HBcrAg (p < 0.05), and preS1 start codon mutation (p < 0.01) were extracted as independent variables. When the influence of the preS codon 132-141deletions on HBsAg and HBcrAg, relative to HBV DNA, was investigated, the HBsAg/HBV DNA ratio was lower (0-10% vs. 10%-100%, p<0.05), while the HBcrAg/HBV DNA rati o was higher (0-0.1% vs. 10%-100%, p<0.05) in the presence of the preS codon 132-141deletions.

Conclusion: The preS codon.132-141 deletions have a significant influence on the clinical characteristics and viral markers, even when present as a minor population. Importantly, the preS codon 132-141 deletions have a clear influence on the viral life cycle and pathogenesis.

Fig 1. HBV preS deletion mapping using deep sequencing.

Deep sequencing of the region of the HBV genome encoding the 174 preS amino acids was carried out and the deleted amino acids were mapped in all 90 patients. Upper panel, the frequency of deletion of each amino acid position in the preS region is expressed for each patient as a color gradient between green and red. In this figure, these sequences are arranged in the order of the FIB-4 values. Lower panel, the average deletion frequency of the preS region per patient is shown.

Fig 2. PreS deletion frequency and its association with clinical factors.

PreS deletion frequency and its association with gender, age and clinical factors (FIB-4 and APRI). A. The patients were stratified into three groups according to their FIB-4 values (over 3.45; n = 30, 1.45–3.45; n = 34, and < 1.45; n = 26). B. The patients were stratified into three groups (5≤; n = 25, 5–1.5; n = 34, and ≤1.5; n = 31) according to their APRI values. C. The patients were divided into two groups according to their sex (male; n = 52, female; n = 38). D. The patients were stratified into two groups according to their age (> 65 y.o., n = 17, ≤ 65 y.o., n = 73).

Fig 3. PreS deletion frequency and its association with viral markers.

The preS deletion frequency and its association with viral markers (HBV DNA, HBcrAg, HBsAg, preS1 start codon mutation, and preS2 start codon mutation). A, The patients were stratified into three groups according to their HBV DNA titers(≥ 6.0 log IU/ml, n = 33, 4.0–6.0 log IU/ml, n = 38 and ≤ 4.0 log IU/ml, n = 19). B. The patients were stratified into three groups according to their HBcrAg titers (> 6.7 log IU/ml, n = 22, 2.9–6.7 log IU/ml, n = 40 and < 2.9 log IU/ml, n = 28). C. The patients were stratified into three groups according to their HBsAg titers (> 3000 IU/m,; n = 19, 1000–3000 IU/ml, n = 27 and < 1000 IU/ml, n = 44). D. The patients were stratified into two groups according to the frequency of preS1 start codon mutations (≥ 1.0%, n = 13 and 0.0–1.0%, n = 77). E. The patients were stratified into two groups according to the frequency of preS2 start codon mutations (≥ 0.5%, n = 55 and 0.0–0.5%, n = 35).

Fig 4. The correlations between the frequency of preS codon 132–141 deletions and host factors and viral markers.

The correlations between the frequency of preS codon 132-141deletions deletions and host factors (age, AFP, and FIB-4) and viral markers (HBV DNA, HBcrAg, genotype and preS1/S2 start codon mutations) are demonstrated.

Fig 5. The impact of preS codon 132–141 deletions on the serum HBsAg and HBcrAg titers, relative to the serum HBV DNA level.

Left panel, the HBsAg titer relative to the HBV DNA value was significantly lower in cases with high frequencies of mutations (10–100%) than in those with low frequencies. Right panel, the HBcrAg titer relative to the HBV DNA value was significantly higher in cases with high frequencies of mutations (10–100%) compared to those with low frequencies (0–0.1%).

Fig 6. Frequency of the preS codon 132–141 deletions and its influence on the requirement for future nucleoside analogue therapy.

Kaplan-Meier curves were drawn according to the frequency of the preS codon 132–141 deletions to determine the influence of the deletions on the requirement for future nucleoside analogue therapy over the ensuing five years. As is shown, compared to the patients with the preS codon 132–141 deletions 0–0.1%, NA therapy was introduced more often in patients with high deletion frequencies (10–100%, p = 0.0095) or with intermediate deletion frequencies (1–10%, p = 0.018).