Association between hepatocellular carcinoma and tumor necrosis factor alpha polymorphisms in South Korea

Table 1 Used primer and restriction enzyme at each polymorphism

Polymorphisms	Forward primer	Reverse primer	Restriction enzyme1
-238 G>A	5’-AGA AGA CCC CCC TCG GAA CC2-3’	5’-ATC TGG AGG AAG CGG TAG TG-3'	MspI
-308 G>A	5’-AGG CAA TAG GTT TTG AGG GCC AT-3'	5’-TCC TCC CTG CTC CGA TTC CG-3'	NcoI
-857 C>T	5′-AAG TCG AGT ATG GGG ACC CCC CGT TAA-3'	5′-CCC CAG TGT GTG GCC ATA TCT TCT T-3'	AseI
-863 C>A	5'-GGC TCT GAG GAA TGG GTT	5'-CTA CAT GGC CCT GTC TTC GTT ACG-3'	MnlI
-1031 T>C	5’-AGC AAG AGC TGT GGG GAG AA-3'	5’-CCT GTA ACC CAT TCC TCA GAG CC-3'	BbsI

¹All of the restriction enzymes were available from New England Biolabs (MA, United States) and we used the reaction conditions recommended by the instructions;

²The underlined bases in the primer were mismatched with the wild-type sequence in order to introduce the restriction enzyme site.

Table 2 Baseline characteristics of study sample n (%)

Variable	Cases	Controls	P value1
n	157	201
Age (yr, mean ± SD)	56.00 ± 11.06	54.00 ± 11.22	0.061
Sex (male, %)	120 (76.4)	148 (73.6)	0.871
Hypertension	19 (12.1)	50 (24.9)	0.012
Diabetes mellitus	30 (19.1)	26 (12.9)	0.195
BMI > 25 kg/m2	40 (25.5)	51 (24.9)	1.000
Smoking	84 (53.5)	78 (38.8)	0.106
Drinking	90 (57.3)	93 (46.3)	0.274
Tumor size
< 5 cm	68 (43.3)	-	-
≥ 5 cm	89 (56.7)	-	-
Portal vein thrombosis
No	92 (58.6)	-	-
Yes	65 (41.4)	-	-
Surgical resection
No	145 (92.4)	-	-
Yes	12 (7.6)	-	-
CTx/RTx
No	27 (17.2)	-	-
Yes	130 (82.8)	-	-
TNM stage
I	34 (21.7)	-	-
II	37 (23.5)	-	-
III	51 (32.5)	-	-
IV	35 (22.3)	-	-
Okuda stage
I	46 (29.3)	-	-
II	68 (43.3)	-	-
III	43 (27.4)	-	-
CTP class
A	83 (52.9)	-	-
B	36 (22.9)	-	-
C	38 (24.2)	-	-

¹Derived from χ² tests for categorical data and Mann-Whitney tests for continuous data. BMI: Body mass index; CTx: Chemotherapy; RTx: Radiotherpay; CTP: Chlid-Turcotte-Pugh.

Table 3 Genotype frequencies and hepatocellular carcinoma adjusted odd ratios for the TNF-α -1031 T>C, -863 C>A, -857 C>T, -308 G>A, and -238 G>A polymorphisms in hepatocellular carcinoma cases and controls n (%)

Characteristics	Controls, n = 201	HCC cases				HCC cases with HBV
		Cases, n = 157	AOR (95%CI)1	P-value	FDR-P-value	Cases, n = 125	AOR (95%CI)1	P-value	FDR-P-value
TNF-α -1031 T>C, rs1799964
TT	121 (60.2)	99 (63.1)	1.000 (reference)			73 (58.4)	1.000 (reference)
TC	70 (34.8)	47 (29.9)	0.796 (0.487-1.301)	0.362	0.586	42 (33.6)	0.922 (0.550-1.544)	0.756	0.960
CC	10 (5.0)	11 (7.0)	1.308 (0.498-3.436)	0.586	0.586	10 (8.0)	1.724 (0.628-4.729)	0.290	0.580
Dominant (TT vs TC + CC)			0.860 (0.540-1.369)	0.524	0.586		1.013 (0.622-1.650)	0.960	0.960
Recessive (TT + TC vs CC)			1.393 (0.540-3.595)	0.493	0.586		1.745 (0.649-4.693)	0.270	0.580
HWE-P	0.976	0.112				0.268
TNF-α -863 C>A, rs1800630
CC	129 (64.2)	98 (62.4)	1.000 (reference)			74 (59.2)	1.000 (reference)
CA	62 (30.8)	53 (33.8)	1.154 (0.709-1.878)	0.565	0.672	46 (36.8)	1.284 (0.767-2.150)	0.341	0.750
AA	10 (5.0)	6 (3.8)	0.784 (0.255-2.414)	0.672	0.672	5 (4.0)	0.825 (0.253-2.690)	0.750	0.750
Dominant (CC vs CA + AA)			1.109 (0.695-1.768)	0.666	0.672		1.226 (0.749-2.007)	0.418	0.750
Recessive (CC + CA vs AA)			0.734 (0.243-2.222)	0.584	0.672		0.760 (0.238-2.428)	0.643	0.750
HWE-P	0.477	0.723				0.513
TNF-α -857 C>T, rs1799724
CC	142 (70.6)	116 (73.9)	1.000 (reference)			92 (73.6)	1.000 (reference)
CT	53 (26.4)	35 (22.3)	0.841 (0.493-1.432)	0.523	0.697	29 (23.2)	0.840 (0.477- 1.478)	0.545	0.893
TT	6 (3.0)	6 (3.8)	1.816 (0.428-7.714)	0.419	0.697	4 (3.2)	1.110 (0.226- 5.438)	0.898	0.893
Dominant (CC vs CT + TT)			0.930 (0.558-1.548)	0.780	0.78		0.888 (0.516-1.531)	0.670	0.893
Recessive (CC + CT vs TT)			2.091 (0.494-8.860)	0.317	0.697		1.500 (0.312-7.203)	0.612	0.893
HWE-P	0.698	0.120				0.371
TNF-α -308 G>A, rs1800629
GG	181 (90.0)	133 (84.7)	1.000 (reference)			108 (86.4)	1.000 (reference)
GA	18 (9.0)	23 (14.6)	1.949 (0.940- 4.040)	0.073	0.178	17 (13.6)	1.779 (0.821-3.853)	0.144	0.430
AA	2 (1.0)	1 (0.6)	0.936 (0.075-11.646)	0.959	0.959	0 (0.0)	N/A	0.995	0.995
Dominant (GG vs GA + AA)			1.841 (0.911-3.720)	0.089	0.178		1.610 (0.758-3.419)	0.215	0.430
Recessive (GG + GA vs AA)			0.861 (0.069-10.734)	0.908	0.959		N/A	0.995	0.995
HWE-P	0.057	0.996				0.415
TNF-α -238 G>A, rs 361525
GG	180 (89.6)	130 (82.8)	1.000 (reference)			104 (83.2)	1.000 (reference)
GA	20 (10.0)	26 (17.6)	1.634 (0.821-3.251)	0.162	0.324	20 (16.0)	1.491 (0.713-3.117)	0.288	0.576
AA	1 (0.5)	1 (0.6)	N/A	0.993	0.994	1 (0.8)	N/A	0.993	0.993
Dominant (GG vs GA + AA)			1.732 (0.876-3.424)	0.115	0.324		1.614 (0.780-3.340)	0.197	0.576
Recessive (GG + GA vs AA)			N/A	0.994	0.994		N/A	0.993	0.993
HWE-P	0.587	0.807				0.972

¹Adjusted for age, sex, hypertension, diabetes mellitus, drinking status, and smoking status. HCC: Hepatocellular carcinoma; AOR: Adjusted odd ratios; HBV: Hepatitis B virus.

Table 4 TNF-α genotype combinations in hepatocellular carcinoma cases and controls¹n (%)

Genotype	HCC cases, n = 157	Controls, n = 201	AOR (95%CI)2	P-value
TNF-α -1031/-857/-238
TT/CC/GG	56 (35.7)	81 (40.3)	1.000 (reference)
TT/CC/GA	11 (7.0)	2 (1.0)	18.849 (2.203-161.246)	0.007
TNF-α -1031/-308/-238
TT/GG/GG	68 (43.3)	103 (51.2)	1.000 (reference)
TT/GG/GA	13 (8.3)	2 (1.0)	26.956 (3.071-236.584)	0.003
TT/GA/GG	17 (10.8)	14 (7.0)	2.712 (1.085-6.778)	0.033
TNF-α -863/-308/-238
CC/GG/GG	68 (43.3)	99 (49.3)	1.000 (reference)
CC/GA/GG	16 (10.2)	13 (6.5)	2.533 (1.007-6.371)	0.048
CA/GG/GA	13 (8.3)	5 (2.5)	4.242 (1.243-14.473)	0.021
TNF-α -1031/-238
TT/GG	86 (54.8)	119 (59.2)	1.000 (reference)
TT/GA	13 (8.3)	2 (1.0)	21.576 (2.581-180.394)	0.005
TNF-α -863/-238
CC/GG	85 (54.1)	114 (56.7)	1.000 (reference)
CA/GA	13 (8.3)	5 (2.5)	3.669 (1.098-12.253)	0.035
TNF-α -308/-238
GG/GG	107 (68.2)	160 (79.6)	1.000 (reference)
GA/GG	23 (14.6)	18 (9.0)	2.283 (1.078-4.836)	0.031
GA+AA/GG	24 (15.3)	20 (10.0)	2.150 (1.041-4.441)	0.039

¹Only combinations significantly associated with HCC are presented in the table;

²Adjusted for age, sex, hypertension, diabetes mellitus, drinking status, and smoking status. HCC: Hepatocellular carcinoma.

Table 5 TNF-α -1031 T>C, -863 C>A, -857 C>T, -308 G>A, and -238 G>A haplotypes in hepatocellular carcinoma cases and controls¹

Haplotype	Overall	Control	Case	OR (95%CI)	P-value
TNF-α -1031/-863/-857/-308/-238
T-C-C-G-G	0.5266	0.5399	0.5139	1.000 (reference)
T-C-C-G-A	0.0119	0.0000	0.0289	25.824 (1.491-447.223)	0.0005
T-C-C-A-G	0.0582	0.0486	0.0647	1.428 (0.749-2.723)	0.320
T-C-T-G-G	0.1334	0.1435	0.1232	0.891 (0.564-1.407)	0.645
T-C-T-A-G	0.0056	0.0040	0.0072	1.360 (0.190-9.764)	1.000
T-A-C-G-G	0.0171	0.0210	0.0000	0.080 (0.005-1.396)	0.023
T-A-C-G-A	0.0109	0.0025	0.0226	4.761 (0.976-23.230)	0.043
T-A-C-A-G	0.0010	0.0000	0.0066	6.796 (0.324-142.621)	0.181
T-A-T-G-G	0.0123	0.0121	0.0133	1.088 (0.288-4.118)	1.000
T-A-T-A-G	0.0009	0.0022	0.0000	0.453 (0.018-11.201)	1.000
C-C-C-G-G	0.0133	0.0103	0.0162	2.176 (0.699-6.778)	0.254
C-C-C-G-A	0.0414	0.0498	0.0318	0.605 (0.257-1.425)	0.306
C-C-T-G-A	0.0042	0.0009	0.0066	6.796 (0.324-142.600)	0.181
C-A-C-G-G	0.1631	0.1638	0.1646	1.088 (0.717-1.652)	0.749
TNF-α -1031/-857/-308/-238
T-C-G-G	0.5430	0.5637	0.5105	1.000 (reference)
T-C-G-A	0.0235	0.0060	0.0531	12.059 (2.747-52.950)	< 0.0001
TNF-α -1031/-857/-238
T-C-G	0.6026	0.6128	0.5854	1.000 (reference)
T-C-A	0.0231	0.0060	0.0517	10.696 (2.428-47.110)	0.0001
TNF-α -1031/-308/-238
T-G-G	0.6885	0.7187	0.6488	1.000 (reference)
T-G-A	0.0238	0.0063	0.0518	7.556 (2.173-26.280)	0.0002
TNF-α -1031/-238
T-G	0.7546	0.7738	0.7297	1.000 (reference)
T-A	0.0233	0.0062	0.0505	10.865 (2.473- 47.740)	0.0001

¹Only data for haplotypes significantly associated with hepatocellular carcinoma are presented in the table. Haplotypes with frequencies of less than zero among cases or controls are not shown.

Table 6 Results of stepwise Cox regression analysis of hepatocellular carcinoma survival

Covariate	β	SEM	HR (95%CI)	P value
Sex	-1.318	0.480	0.268 (0.105-0.682)	0.006
Chemotherapy or radiotherapy	-1.914	0.500	0.148 (0.056-0.391)	0.0001
Portal vein thrombosis	1.334	0.478	3.795 (1.495-9.631)	0.005
TNF-α -1031 TT vs CC	1.772	0.733	5.881 (1.408-24.554)	0.016

Table 7 TNF-α genotypes and survival in Okuda stage III hepatocellular carcinoma cases

Genotype	Validation set		Adjusted HR (95%CI)1	P-value
	Cases (n = 43)	Deaths (n = 41)
TNF-α -1031 T>C, rs1799964
TT	29 (67.4)	27 (65.9)	1.000 (reference)
TC	11 (25.6)	11 (26.8)	1.314 (0.448-3.856)	0.621
CC	3 (7.0)	3 (7.3)	5.795 (1.145-29.323)	0.035
Dorminant model (TT vs TC + CC)			1.805 (0.733-4.442)	0.201
Recessive model (TT + TC vs CC)			3.761 (0.869-16.265)	0.078

¹Adjusted for age, sex, hypertension, diabetes mellitus, drinking status, smoking status, portal vein thrombosis, tumor size, surgical resection, and chemotherapy or radiotherapy.