Prevalence of Potential Resistance Related Variants Among Chinese Chronic Hepatitis B Patients Not Receiving Nucleos(T)ide Analogues

Figures & data

Table 1 Demographic and Clinical Characteristics of CHB Patients

Characteristics	HBeAg + (n=119)	HBeAg – (n=87)	P
Age(years), (mean±SD)	32.7±12.2	42.3±11.2	<0.001
Gender(male/female)	85/34	60/27	0.702
HBV-DNA(log 10IU/mL), (mean±SD)	6.9±1.5	5.2±1.4	<0.001
HBV genotype (B/C)	61/58	60/27	0.011
ALT(IU/L), median(range)	76.8(15.0–2083.9)	54.5(15.5–526.8)	0.051
AST(IU/L), median(range)	50.8(13.7–1883.1)	41.0(14.7–357.1)	0.289
ALP(IU/L), median(range)	89.5(18.0–321.0)	87.7(42.4–221.6)	0.865
GGT(IU/L), median(range)	30.0(7.0–662.5)	36.0(6.0–495.1)	0.237
TBil(IU/L), median(range)	18.3(4.6–226.4)	18.6(6.8–249.6)	0.302

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, γ-glutamyl transpeptidase; TBil, total bilirubin; SD, standard deviation.

Table 2 Genotype-Dependent AA Polymorphic Sites in This Study

Genotype	rt53						rt91		rt124
	N	S	A^a	D^a	H^a	I^a	I	L	N	Y	D^a	H^a
B (n=121)	112	1	1	5	1	1	3	118	109	2	9	1
C (n=85)	0	85	0	0	0	0	77	8	0	78	0	7
P	<0.001						<0.001		<0.001
Genotype	rt134					rt221
	D	N	A^a	E^a	H^a	S^a	F	Y	C^a	H^a	N^a
B (n=121)	22	92	0	1	0	6	0	118	1	1	1
C (n=85)	77	3	1	3	1	0	83	2	0	0	0
P	<0.001						<0.001
Genotype	rt224		rt238			rt256
	I	V	H	N	D^a	C	S	G^a
B (n=121)	4	117	119	2	0	0	110	11
C (n=85)	79	6	1	82	2	12	73
P	<0.001		<0.001			<0.001

Notes: ^aDescribed as RT variants in this study.

Abbreviations: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine H, histidine; I, isoleucine; L, leucine; N, asparagine; S, serine; V, valine; Y, tyrosine.

Table 3 Prevalence of Potential DR-Related Variants of RT Region in CHB Patients

Mutation Category	Type of Variants	Genotype B (n=121)	Genotype C (n=85)	P
		Number of Mutations (%)
Primary resistance variant	I169T	2 (1.6)	1 (1.2)	0.779
	A181V/T	1 (0.8)	2 (2.4)	0.570
	M204I/V	4 (3.2)	4 (4.8)	0.720
	N236T	1 (0.8)	1 (1.2)	1.000
Secondary/compensatory variant	L80I/V	3 (2.4)	2 (2.4)	1.000
	V173L	0 (0.0)	2 (2.4)	0.169
	L180M	1 (0.8)	3 (3.6)	0.308
Putative DR variant	N53A/D/H/I	8 (6.4)	0 (0.0)	0.022
	L82Q	1 (0.8)	0 (0.0)	1.000
	V84I	0 (0.0)	1 (1.2)	0.413
	H126Q/Y	0 (0.0)	2 (2.4)	0.169
	T128A/D/I/N/P	5 (4.0)	6 (7.2)	0.367
	N139D	0 (0.0)	1 (1.2)	0.413
	R153Q	0 (0.0)	1 (1.2)	0.413
	V191I	0 (0.0)	2 (2.4)	0.169
	V207I/L/M	5 (6.0)	3 (3.6)	1.000
	S213T	7 (5.6)	2 (2.4)	0.312
	V214A	0 (0.0)	1 (1.2)	0.413
	Q215H	2 (1.6)	1 (1.2)	0.779
	Y221C/N/H	3 (2.4)	0 (0.0)	0.269
	L229V/M	3 (2.4)	2 (2.4)	1.000
	I233V	1 (0.8)	0 (0.0)	1.000
	P237S	1 (0.8)	0 (0.0)	1.000
	N238D	0 (0.0)	2 (2.4)	0.169
	S/C256G	11 (8.8)	0 (0.0)	0.003
Pretreatment variant	T38A	0 (0.0)	1 (1.2)	0.413
	N/Y124D/H	10 (8.0)	7 (8.4)	0.994
	D/N134A/E/H/S	7 (5.6)	5 (6.0)	1.000
	N139K	2 (1.6)	3 (3.6)	0.405
	R242S	0 (0.0)	1 (1.2)	0.413

Figure 1 RT variants in genotype B and C HBV infected patients.

Abbreviations: RT, reverse transcriptase; CHB, chronic hepatitis B; HBV, hepatitis B virus.

Table 4 Variation Distribution and Frequency in Different RT Regions

Region in RT(Sites Studied in This Study)	Number of Variation Sites (%)		P	Frequency (%)	P
Domain(22)	Genotype B	11/22(50)	1.000^a	31/4532 (0. 68%)	0.215^a
	Genotype C	11/22(50)		22/4532 (0.48%)
Total	15/22(68.2)		0.288^b	53/4532 (1.17%)	<0.001^c
A-B interdomain(6)	Genotype B	4/6(66.7)	0.455^a	24/1236 (1.94%)	0.885^a
	Genotype C	6/6(100)		25/1236 (2.02%)
Total	6/6(100.0)		0.115^b	49/1236 (3.96%)	<0.001^c
Non-A-B interdomain (14)	Genotype B	5/14(35.7)	0.699^a	9/2884(0.31%)	0.013^a
	Genotype C	6/14(42.8)		23/2884 (0.79%)
Total	8/14(57.1)		0.501^b	32/2884(1.11%)	0.813^c

Notes: ^aComparison of number of variation sites and frequency in different RT sections between B-genotype and C genotype. ^b Comparison of number of variation sites among different RT region were as follow: Domain vs A-B interdomain, P=0.288; Domain vs Non-A-B interdomain, P=0.501; A-B interdomain vs Non-A-B interdomain, P=0.115. ^c Comparison of variation frequency among different RT region were as follow: Domain vs A-B interdomain, P<0.001; Domain vs Non-A-B interdomain, P=0.813; A-B interdomain vs Non-A-B interdomain, P<0.001. For example, the calculation method of variation frequency was as follows: 31 mutations/(22 studies sites×206 isolates)*100%=31/4532×100%=0.68%

Table 5 Clinical Features of CHB Patients with and without Potential DR-Related Variants

Characteristics	Patients without Variants (n=118)	Patients with Variants (n=88)	P
Gender(male/female)	80/38	65/23	0.345
Age(years), (mean±SD)	35.2±12.9	38.9±12.1	0.014
HBeAg status(+/-)	75/43	44/44	0.051
HBV DNA(log 10IU/mL), (mean±SD)	6.47±1.68	5.84±1.63	0.007
HBV genotype(B/C)	70/48	51/37	0.884
ALT(IU/L), median(range)	81.4(15.0–2083.9)	52.8(18.2–992.8)	0.041
AST(IU/L), median(range)	52.7(14.7–1883.1)	39.3(13.7–719.0)	0.070
ALP(IU/L), median(range)	92.5(30–220.9)	83.3(18.0–321.0)	0.154
GGT(IU/L), median(range)	30.2(6.0–495.1)	31.8(7.0–662.5)	0.527
TBil(IU/L), median(range)	18.4(4.6–226.4)	18.6(6.3–249.6)	0.728

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, γ-glutamyl transpeptidase; TBil, total bilirubin; SD, standard deviation.

Figure 2 HBV-DNA loads in untreated CHB patients with or without RT variants. (A) All patients (B) Patients with HBeAg positive (C) Patients with HBeAg negative.

Abbreviations: HBV, hepatitis B virus; CHB, chronic hepatitis B; RT, reverse transcriptase; HBeAg, hepatitis B virus e antigen.

Table 6 Comparison of Clinical Characteristics in CHB Patients with Different Number of Potential DR-Related Variants

Characteristics	Number of RT Variants		P
	Single Variant	Multiple Variants (≥2)
Gender(male/female)	44/12	21/11	0.184
Age (years)	39.1±12.3	38.5±12.1	0.827
HBeAg status(+/-)	24/32	20/12	0.076
HBV DNA(log 10IU/mL), (mean±SD)	5.85±1.74	5.81±1.43	0.922
HBV genotype(B/C)	33/23	18/14	0.807
ALT(IU/L), median(range)	52.5(18.6–992.8)	44.2(18.2–887.5)	0.195
AST(IU/L), median(range)	41.4(13.7–636.9)	35.8(14.8–719.0)	0.149
ALP(IU/L), median(range)	84.8(18.0–221.6)	82.8(46.9–321.0)	0.859
GGT(IU/L), median(range)	32.8(7.0–662.5)	31.5(8.8–191.1)	0.374
TBil, (IU/L), median(range)	18.4(7.2–157.9)	19.6(6.3–249.6)	0.469

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, γ-glutamyl transpeptidase; TBil, total bilirubin; SD, standard deviation.

Table 7 Univariate and Multivariate Logistic Analyses of Factors for Potential DR-Related Variants

Factor		Univariate Analysis				Multivariate Analysis
		OR	95% CI		P	OR	95% CI		P
			Lower	Upper			Lower	Upper
Age	<35yr	1				1
	≥35yr	1.024	1.001	1.047	0.037	1.661	0.886	3.112	0.114
HBeAg	Negative	1				1
	Positive	0.573	0.327	1.005	0.052	0.916	0.470	1.788	0.798
HBV DNA	<10^6IU/mL	1				1
	≥10^6IU/mL	0.794	0.670	0.941	0.008	0.452	0.257	0.794	0.006
ALT	<80IU/L	1				1
	≥80 IU/L	0.999	0.997	1.000	0.105	0.706	0.391	1.272	0.246

Note: P-value by logistic regression analysis.

Abbreviations: OR, odds ratio; CI, confidence interval.