Deep sequencing analysis of hepatitis B virus mutations in the basal core promoter and precore regions

doi:10.3877/cma.j.issn.1674-1358.2016.02.005

Abstract

Abstract:

Objective

To explore the characteristics of mutations in the basal core promoter (BCP) and precore (PC) regions of hepatitis B virus (HBV) genome in HBeAg-positive patients with chronic hepatitis B (CHB) using Ion Torrent Personal Genome Machine (PGM) deep sequencing.

Methods

Serum samples from 25 HBeAg-positive patients with CHB were collected, which were used for HBV DNA extraction. Nested PCR was used to amplify the BCP and PC regions of HBV genome. The PCR product was subjected to deep sequencing libraries preparation and subsequently Ion Torrent PGM sequencing. The variants and mutant percentages were detected through bioinformatics analysis. HBV BCP/PC wild type and mutant type reference plasmids were constructed, which were used as quality control in deep sequencing.

Results

Ten single nucleotide polymorphisms (SNPs) that had variant types with prevalence of greater than 20% were observed in the BCP and PC regions among the 25 HBeAg-positive patients with CHB: G1746A, A1752G/T, T1753C/G, A1762T, G1764A, C1817G/A, T1825C/A, A1846T, G1896A and G1899A; the prevalence of G1746A and T1825C/A were 92% and 100%, respectively. The prevalence of these mutants was analyzed in patients with different HBV genotype infection. The A1752G/T and G1896A were mainly prevalent in genotype B infection HCV patients (63.6% vs 0.0%, χ² = 12.374, P = 0.0007 and 72.7% vs 28.6%; χ² = 4.812, P = 0.0472), A1762T and G1764A were mainly prevalent in genotype C HCV infection patients (27.3% vs 78.6%; χ² = 6.579, P = 0.0172). Patients with genotype C HCV infection had higher A1762T/G1764A mutant percentages than those with genotype B HCV infection, but without statistical significance (25.7% ± 28.4% vs 68.4% ± 42.7%; t = 1.614, P = 0.1326). Among the 25 CHB patients with HBeAg-positive, 32.0% cases had BCP mutants only, 24.0% had PC mutant only, and 24.0% had both BCP and PC mutants.

Conclusions

Deep sequencing could be used for quantification of HBV BCP and PC mutants, providing a suitable technology platform for the clinical application of HBV mutant research.

Key words: Hepatitis B virus, Basal core promoter, Precore, Deep sequencing

Linlin Yan, Henghui Zhang, Hui Ma, Di Liu, Wei Li, Hongsong Chen, Qixiang Shao. Deep sequencing analysis of hepatitis B virus mutations in the basal core promoter and precore regions[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2016, 10(02): 151-156.

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References 25

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特征		CHB （n = 25）
年龄（±s，岁）		30.2 ± 8.6
性别[例（%）]
	女	5（20.0）
	男	20（80.0）
HBV基因型[例（%）]
	B型	11（44.0）
	C型	14（56.0）
抗-HBe [例（%）]
	阳性	0（0.0）
	阴性	25（100.0）
血清HBV DNA水平[例（%）]
	10⁵～10⁸拷贝/ml	18（72.0）
	≥ 10⁸拷贝/ml	7（28.0）
血清HBsAg水平[例（%）]
	100～10 000 IU/ml	6（24.0）
	≥ 10 000 IU/ml	19（76.0）
ALT（±s，U/L）		174.3 ± 75.9
AST（±s，U/L）		86.9 ± 62.6

特征		CHB （n = 25）
年龄（±s，岁）		30.2 ± 8.6
性别[例（%）]
	女	5（20.0）
	男	20（80.0）
HBV基因型[例（%）]
	B型	11（44.0）
	C型	14（56.0）
抗-HBe [例（%）]
	阳性	0（0.0）
	阴性	25（100.0）
血清HBV DNA水平[例（%）]
	10⁵～10⁸拷贝/ml	18（72.0）
	≥ 10⁸拷贝/ml	7（28.0）
血清HBsAg水平[例（%）]
	100～10 000 IU/ml	6（24.0）
	≥ 10 000 IU/ml	19（76.0）
ALT（±s，U/L）		174.3 ± 75.9
AST（±s，U/L）		86.9 ± 62.6

区域	突变位点	例（%）	突变率（±s，%）	C基因型（n = 14）	B基因型（n = 11）
BCP（1 742-1 849）	G1746A	23（92.0）	14.4 ± 6.6	13（92.9）	10（90.9）
	A1752G/T	7（28.0）	85.4 ± 29.9	0（0.0）	7（63.6）
	T1753C/G	6（24.0）	8.9 ± 7.6	5（35.7）	1（9.1）
	A1762T	14（56.0）	59.3 ± 43.1	11（78.6）	3（27.3）
	G1764A	14（56.0）	47.4 ± 43.3	11（78.6）	3（27.3）
PC（1 814-1 900）	C1817G/A	6（24.0）	2.7 ± 1.1	1（7.1）	5（45.5）
	T1825C/A	25（100.0）	27.6 ± 11.5	14（100）	11（100）
	A1846T	7（28.0）	21.7 ± 21.6	2（14.3）	5（45.5）
	G1896A	12（48.0）	10.7 ± 16.6	4（28.6）	8（72.7）
	G1899A	5（20.0）	5.9 ± 8.1	4（28.6）	1（9.1）

区域	突变位点	例（%）	突变率（±s，%）	C基因型（n = 14）	B基因型（n = 11）
BCP（1 742-1 849）	G1746A	23（92.0）	14.4 ± 6.6	13（92.9）	10（90.9）
	A1752G/T	7（28.0）	85.4 ± 29.9	0（0.0）	7（63.6）
	T1753C/G	6（24.0）	8.9 ± 7.6	5（35.7）	1（9.1）
	A1762T	14（56.0）	59.3 ± 43.1	11（78.6）	3（27.3）
	G1764A	14（56.0）	47.4 ± 43.3	11（78.6）	3（27.3）
PC（1 814-1 900）	C1817G/A	6（24.0）	2.7 ± 1.1	1（7.1）	5（45.5）
	T1825C/A	25（100.0）	27.6 ± 11.5	14（100）	11（100）
	A1846T	7（28.0）	21.7 ± 21.6	2（14.3）	5（45.5）
	G1896A	12（48.0）	10.7 ± 16.6	4（28.6）	8（72.7）
	G1899A	5（20.0）	5.9 ± 8.1	4（28.6）	1（9.1）

突变位点		例（%）	突变率（%，±s）	P值
BCP A1762T				0.1326
	B基因型	3（27.3）	25.7 ± 28.4
	C基因型	11（78.6）	68.4 ± 42.7
PC G1896A				0.4995
	B基因型	8（72.7）	13.2 ± 19.6
	C基因型	4（28.6）	5.9 ± 8.0
BCP/PC组合				0.1869
	W/W	5（20.0）	0/0
	W/M	6（24.0）	0/14.9 ± 22.9
	M/W	8（32.0）	88.6 ± 27.5/0
	M/M	6（24.0）	20.2 ± 23.6/6.5 ± 5.8

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