Association between polymorphisms of the APOBEC3G gene and chronic hepatitis B viral infection and hepatitis B virus-related hepatocellular carcinoma

Table 1 Primer sequences and reaction conditions for genotyping APOBEC3G polymorphisms

SNP	Sequences of the primers	Annealing temperature, °C
Rs7291971	F: 5’-ACGTTGGATGGATCATCTGAGGTCAGTGTC-3’	59.6
	R: 5’-ACGTTGGATGCCATCTGGATGTATATGTGC-3’
Rs5757465	F: 5’-ACGTTGGATGTGTACAAGGGATATGGCCAC-3’	54.6
	R: 5’-ACGTTGGATGAATCTGGGTCCCAGAAGTAG-3’
Rs5757463	F: 5’-ACGTTGGATGTAATTTGTAGGTCACCACGC-3’	50.9
	R: 5’-ACGTTGGATGAGCCTGTCTGGAGCCTCCCT-3’
Rs8177832	F: 5’-ACGTTGGATGGAGCCTTGGAATAATCTGCC-3’	51.5
	R: 5’-ACGTTGGATGGAGACCCTCACCTGAGAATC-3’
Rs2011861	F: 5’-ACGTTGGATGTCTTTTCCCGCAGGATGAAG-3’	55.7
	R: 5’-ACGTTGGATGATTTGAGGATCAGGGCCTAC-3’

Table 2 Baseline characteristics of study subjects, n (%)

Group	Healthy controls,	Chronic HBV infection patients				P value3
	n = 299	Non-HCC		HBV-related HCC
		n = 370	P value1	n = 287	P value2
Male	247 (82.6)	295 (79.7)	0.345	246 (85.7)	0.304	0.921
Age, M (P25, P75)	50 (45, 55)	49 (42, 55)	0.078	50 (46, 56)	0.589	0.408
Smoking			0.392		0.003	0.292
Ever	113 (37.8)	128 (34.6)		144 (50.2)
Never	186 (62.2)	242 (65.4)		143 (49.8)
Drinking			0.036		0.804	0.241
Ever	122 (40.8)	122 (33.0)		120 (41.8)
Never	177 (59.2)	248 (67.0)		167 (58.2)

P values represent the non-HCC¹, HCC², and chronic HBV³ patients compared to the healthy controls. HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma.

Table 3 Genotype and allele frequencies of single nucleotide polymorphisms in the APOBEC3G gene in each group

SNP	Healthy controls	Chronic HBV infection patients						Hepatitis B patients (n = 657) vs healthy controls (n = 299)
		Non-HCC, n = 370			HBV-related HCC, n = 287
	n = 299 (%)	n (%)	OR (95%CI)	P value1	n (%)	OR (95%CI)	P value2	OR (95%CI)	P value3
Rs7291971 genotype and allele
Detected number	n = 290	n = 366			n = 282			n = 648 vs n = 290
CC	28 (9.7)	36 (9.8)	1		26 (9.2)	1		1
CG	113 (39.0)	139 (38.0)	0.92 (0.53-1.61)	0.778	114 (40.4)	1.14 (0.63-2.09)	0.667	1.00 (0.61-1.66)	0.987
GG	149 (51.4)	191 (52.2)	0.98 (0.57-1.68)	0.935	142 (50.4)	1.14 (0.63-2.06)	0.664	1.04 (0.64-1.69)	0.891
CG + GG	262	330	0.95 (0.57-1.61)	0.867	256	1.14 (0.65-2.02)	0.651	1.02 (0.64-1.64)	0.930
C allele	169 (29.1)	211 (28.8)	1		166 (29.4)	1		1
G allele	411 (70.9)	521 (71.2)	1.02 (0.80-1.29)	0.901	398 (29.1)	0.99 (0.76-1.27)	0.913	1.00 (0.80-1.24)	0.983
Rs5757463 genotype and allele
Detected number	n = 285	n = 369			n = 285			n = 654 vs n = 285
CC	241 (84.6)	308 (83.5)	1		227 (79.6)	1		1
CG	43 (15.1)	56 (15.2)	1.02 (0.66-1.57)	0.944	53 (18.6)	1.34 (0.86-2.10)	0.202	1.16 (0.79-1.71)	0.456
GG	1 (0.4)	5 (1.4)	1.93 (0.37-10.14)	0.439	5 (1.8)	2.71 (0.51-14.31)	0.239	2.25 (0.49-10.39)	0.298
CG + GG	44	61	1.06 (0.69-1.62)	0.799	58	1.40 (0.91-2.17)	0.130	1.21 (0.83-1.77)	0.327
C allele	525 (92.1)	672 (91.1)	1		507 (88.9)	1		1
G allele	45 (7.9)	66 (8.9)	1.15 (0.77-1.70)	0.500	63 (11.1)	1.45 (0.97-2.17)	0.069	1.28 (0.90-1.82)	0.194
Rs5757465 genotype and allele
Detected number	n = 285	n = 365			n = 279			n = 644 vs n = 285
TT	170 (59.6)	221 (60.5)	1		169 (60.6)	1		1
TC	101 (35.4)	129 (35.3)	1.02 (0.73-1.42)	0.898	92 (33.0)	0.93 (0.65-1.34)	0.707	0.98 (0.73-1.32)	0.903
CC	14 (4.9)	15 (4.1)	0.82 (0.38-1.76)	0.610	18 (6.5)	1.43 (0.68-3.01)	0.349	1.05 (0.55-2.02	0.884
TC + CC	115	144	0.99 (0.72-1.37)	0.984	110	0.99 (0.70-1.39)	0.953	0.99 (0.74-1.32	0.945
T allele	441 (77.4)	571 (78.2)	1		430 (77.1)	1		1
C allele	129 (22.6)	159 (21.8)	0.95 (0.73-1.24)	0.714	128 (22.9)	1.02 (0.77-1.34)	0.902	0.98 (0.77-1.24)	0.868
Rs8177832 genotype and allele
Detected number	n = 291	n = 369			n = 287			n = 656 vs n = 291
AA	227 (78.0)	302 (81.8)	1		249 (86.8)	1		1
AG	60 (20.6)	65 (17.6)	0.80 (0.54-1.18)	0.253	35 (12.2)	0.53 (0.33-0.84)	0.007	0.67 (0.47-0.96)	0.029
GG	4 (1.4)	2 (0.5)	0.40 (0.72-2.26)	0.302	3 (1.0)	0.75 (0.16-3.43)	0.711	0.55 (0.15-2.07)	0.374
AG + GG	64	67	0.78 (0.53-1.14)	0.195	38	0.54 (0.35-0.85)	0.007	0.66 (0.47-0.94)	0.021
A allele	514 (88.3)	669 (90.7)	1		533 (92.9)	1		1
G allele	68 (11.7)	69 (9.3)	0.78 (0.55-1.11)	0.167	41 (7.1)	0.58 (0.39-0.87)	0.008	0.69 (0.50-0.95)	0.023
Rs2011861 genotype and allele
Detected number	n = 284	n = 364			n = 279			n = 643 vs n = 284
CC	53 (18.7)	55 (15.1)	1		33 (11.8)	1		1
CT	127 (44.7)	170 (46.7)	1.25 (0.80-1.95)	0.328	135 (48.4)	1.69 (1.02-2.80)	0.042	1.42 (0.95-2.13)	0.085
TT	104 (36.6)	139 (38.2)	1.27 (0.81-2.02)	0.301	111 (39.8)	1.82 (1.08-3.06)	0.024	1.48 (0.98-2.24)	0.063
CT + TT	231	309	1.26 (0.83-1.91)	0.277	246	1.75 (1.08-2.82)	0.022	1.45 (0.99-2.11)	0.053
C allele	233 (41.0)	280 (38.5)	1		201 (36.0)	1		1
T allele	335 (59.0)	448 (61.5)	1.11 (0.89-1.39)	0.350	357 (64.0)	1.24 (0.97-1.57)	0.085	1.16 (0.95-1.42)	0.140

P values represent the non-HCC¹, HCC², and chronic HBV³ infection patients compared to the healthy control groups adjusted for age, sex, smoking, and drinking by logistic regression analysis. The two-sided χ² test or Fisher’s exact test was used in alleles distribution comparison. HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma.

Table 4 Haplotype distributions between healthy controls and chronic hepatitis B virus infection patients, n (%)

Haplotype	Frequency		χ2	P value	OR (95%CI)
	Healthy controls	Chronic HBV infection patients
rs5757463-rs8177832
C-A	455 (80.3)	1067 (81.7)	6.07	0.048	1
C-G	66 (11.7)	110 (8.4)			0.71 (0.51-0.98)
G-A	45 (8.0)	129 (9.9)			1.22 (0.86-1.75)

HBV: Hepatitis B virus.

Table 5 Haplotype distributions between healthy controls and hepatocellular carcinoma patients, n (%)

Haplotype	Frequency		χ2	P value	OR (95%CI)
	Healthy controls	HCC
rs7291971-rs8177832
C-A	166 (29.0)	166 (29.4)	6.73	0.03	1
G-A	339 (59.3)	357 (63.3)			1.05 (0.81-1.37)
G-G	67 (11.7)	41 (7.3)			0.61 (0.39-0.95)
rs5757463-rs8177832
C-A	455 (80.4)	466 (81.8)	8.96	0.01	1
C-G	66 (11.7)	41 (7.2)			0.61 (0.40-0.91)
G-A	45 (8.0)	63 (11.1)			1.37 (0.91-2.05)
rs5757465-rs8177832
T-A	371 (66.0)	390 (69.9)	6.94	0.03	1
C-A	125 (22.2)	128 (22.9)			0.97 (0.73-1.30)
T-G	66 (11.7)	40 (7.2)			0.58 (0.38-0.88)
rs5757463-rs2011861
C-C	183 (33.2)	141 (25.4)	10.68	0.01	1
C-T	326 (59.1)	354 (63.7)			1.41 (1.08-1.83)
G-C	43 (7.8)	60 (10.8)			1.80 (1.15-2.82)
G-T	0 (0.0)	1 (0.2)			1.00 (0.99-1.02)
rs7291971-rs5757463-rs8177832
C-C-A	118 (21.1)	101 (18.0)	10.59	0.01	1
C-G-A	45 (8.0)	63 (11.3)			1.64 (1.03-2.61)
G-C-A	332 (59.3)	355 (63.4)			1.25 (0.92-1.70)
G-C-G	65 (11.6)	41 (7.3)			0.74 (0.46-1.18)
rs7291971-rs5757463-rs2011861
C-C-C	112 (20.4)	91 (16.6)	11.62	0.04	1
C-C-T	5 (0.9)	6 (1.1)			1.63 (0.42-6.42)
C-G-C	43 (7.8)	59 (10.8)			1.70 (1.05-2.76)
C-G-T	0 (0.0)	2 (0.4)			1.02 (0.99-1.05)
G-C-C	70 (12.8)	48(8.8)			0.85 (0.85-1.35)
G-C-T	319 (58.1)	342 (62.4)			1.33 (0.97-1.82)

HCC: Hepatocellular carcinoma.