Naturally occurring mutations in the reverse transcriptase region of hepatitis B virus polymerase from treatment-naïve Korean patients infected with genotype C2

Table 1 Comparison of clinical data between chronic hepatitis and hepatocellular carcinoma patients

Clinical factors	CH (n = 59)	HCC (n = 72)	Total (n = 131)	P value
Age, yr, mean ± SD	38.9 ± 11.1	52.3 ± 9.7	45.7 ± 12.3	< 0.001
Gender, male	47.40%	79.10%	64.80%	< 0.001
HBeAg negative	35.50%	54.10%	45.80%	0.04%
ALT (IU/L), mean ± SD	94.5 ± 105.6	74.2 ± 85.1	106.8 ± 191.2	NS
AST (IU/L), mean ± SD	70.4 ± 92.0	127.1 ± 139.8	113.2 ± 141.0	< 0.001
HBV DNA	6.5 ± 2.0	5.3 ± 1.1	6.53 ± 1.7	< 0.001
HBsAg	3.7 ± 0.6	3.3 ± 0.7	3.43 ± 0.6	< 0.001

HbsAg: Hepatitis B virus surface antigen.

Table 2 Correlation between the frequency of potential nucleos(t)ide analog resistance mutation and clinical features

No. of mutations	CH/HCC	HBeAg (positive/negative)	ALT (IU/L)	AST (IU/L)	HBV DNA	HBsAg
0	27/25	29/23	108.51 ± 105.8	122.92 ± 106.8	6.50 ± 6.5	3.97 ± 3.8
1	18/16	21/13	101.61 ± 108.7	71.42 ± 126.1a	6.91 ± 6.4	3.95 ± 3.8
2	9/19	15/13	81.10 ± 113.8	113.75 ± 113.0	6.65 ± 6.1	3.65 ± 3.9a
3	4/7	3/8	211.45 ± 97.3	205.18 ± 104.8a	6.84 ± 6.5	3.84 ± 3.8
4	1/4	2/3	56.60 ± 108.8	72.00 ± 114.8	5.85 ± 6.5	3.68 ± 3.8
5	0/1	1/0	24.23	39.05	7.49	2.83
≥ 1 (n = 79)	32/47	42/37	105.81 ± 216.19	106.86 ± 131.51	5.11 ± 1.55	3.37 ± 0.77
Total (n = 131)	59/72	71/60	107.21 ± 191.16	113.2 ± 141.06	4.98 ± 1.51	3.43 ± 0.73

The significant values were shown in boldface and marked with asterisk (^aP < 0.05). HbsAg: Hepatitis B virus surface antigen; HCC: Hepatocellular carcinoma.

Table 3 Characterization of potential 42 NAr mutation from treatment naive Korean patients of genotype C2 infections

Category	Mutation	Drug resistance	CH	HCC	P value
Primary drug resistance	T184A/C/F	ETV	1	-	0.021
	M204I/V	LMV, ETV, TNF	-	9
Mutation number (%)/no. of patients number			1/472 (0.21%)	9/576 (1.56%)
			1	9 patients
Secondary mutation	L80I	LMV	-	5	NS
	L180M	LMV, ETV, LdT	1	2
Mutation number (%)/no. of patients number			1/177 (0.56%)	7/216 (3.24%)
			1	6
Putative NAr mutation	S53N	LMV	1	1	N.S
	L82M/V	LMV	-	1
	V84M/I	ADV	1	-
	H126C/Y/Q	ADV	5	6
	I128I/N/A	LMV	11	5
	R/W153Q	LMV	2	-
	V191I/D	LMV, ADV	2	3
	V207I	LMV	-	1
	S213T	ADV	-	3
	Q215P/S/H	LMV, ADV	-	2
	L217R	ADV	1	-
	F221Y	ADV	3	9
	L229G/V/W	LMV	-	2
	P237H	ADV	-	2
	N238D/S/T	ADV	3	6
Mutation number (%)/no. of patients number			29/1475 (1.96%)	41/1800 (2.27%)
			23	31
Pre-treatment mutation	T38A		9	5	NS
	Y124H	Found	4	6
	D134E/N/C	Before	4	8
	N139K/H	Therapy	-	8
	I224V		4	12
Mutation number (%)/no. of patients number			21/351 (5.93%)	39/432 (9.02%)
			17	31
Total Mutation number (%)/no. of patients (%)			52/2478 (2.09)	96/3024 (3.17)	0.003
			32 patients (54.2)	47 patients (65.2)
			148/5502 (2.68)
			79 (60.3)

ETV: Entecavir; LMV: Lamivudine; TNF: Tenofovir; ADV: Adefovir; CH: Chronic hepatitis; HCC: Hepatocellular carcinoma.

Table 4 Mutation site distributions and mutation rate in different sections of hepatitis B virus RT and overlapped hepatitis B surface antigen regions

	Region (amino acid)	No. of mutation site	Mutation frequency	P value
Reverse transcriptase	Domain (22)	9 (40.9)	1.07%	0.008
	A-B interdomain (6)	6 (100)	7.50%	-
	Non A-B interdomain (14)	10 (71.4)	3.16%	NS
	Total (42)	25 (59.5)	2.68%	-
HBsAg	A-determinant (3)	2 (66.6)	3.81%
	Non A-determinant (37)	8 (21.6)	0.55%	< 0.001
	Total (40)	10 (28.5)	0.80%	-

Statistics were calculated between Domain/Non A-B interdomain and A-B interdomain in reverse transcriptase region. In category 2, the statistical significant showed between A-determinant and Non-A-determinant in HBsAg region.

Table 5 Frequency and patterns of 3 types of NAr Mutations related to hepatocellular carcinoma

Mutations	No. of patients		Nucleotide sequences	Codons in RT genes (patients)	Codons in HBsAg genes (patients)	P value
	CH	HCC
rtL80I	0	5	GGCTAT→GGATAT	CTA(L)→ATA(I) (5)	TAT(Y)→TAT(Y) (5)	0.036
rtN139K/T/H (sT131N/P)	0	8	GGAACC→GGAAAC	AAC(N)→AAA(K) (4)	ACC(T)→AAC(N) (4)
			→GGCACC	→CAC(H) (3)	→ACC(T) (3)	0.008
			→GGACCC	→ACC(T) (1)	→CCC(P) (1)
rtM204I/V (sW196L/S/W)	0	9	ATATGG→ATATTG	ATG(M)→ATT(I) (7)	TGG(W)→TTG(L) (7)
			→ATATCG	→ATC(I) (1)	→TCG(S) (1)	0.004
			→ATGTGG	→GTG(V) (1)	→TGG(W) (1)

The point mutation bases of the three truncations are shown in bold and italic. H: Histidine; I: Isoleucine; K: Lysine; L: Leucine; M: Methionine; N: Asparagine; P: Proline; S: Serine; T: Threonine; V: Valine; W: Tryptophan; Y: Tyrosine; CH: Chronic hepatitis; HCC: Hepatocellular carcinoma.

Table 6 Comparison of clinical features between patients with or without L80I

Clinical factors	Wild type (n = 126)	L80I (n = 5)	Total (n = 131)	P value
Age, yr, mean ± SD	45.8 ± 12.2	57.2 ± 8.1	45.7 ± 12.3	0.043
Gender, Male	63.50%	100%	64.80%	NS
HBeAg negative	45.20%	60.00%	45.80%	NS
ALT (IU/L), mean ± SD	84.0 ± 96.8	68.6 ± 19.7	100.8 ± 191.2	NS
AST (IU/L), mean ± SD	100.9 ± 125.3	118.6 ± 65.0	113.2 ± 141.0	NS
HBV DNA	5.8 ± 1.7	6.7 ± 0.2	6.5 ± 1.7	< 0.001
HBsAg	3.4 ± 0.65	3.5 ± 0.32	3.4 ± 0.6	NS
CH: HCC, HCC (%)	59/67 (53.9)	0/5 (100)	59/72 (54.9)	0.036

HBV: Hepatitis B virus; CH: Chronic hepatitis; HCC: Hepatocellular carcinoma.