Polymorphisms of the TLR2 and TLR4 genes are associated with risk of gastric cancer in a Brazilian population

Table 1 Primer sequences, restriction enzymes and fragment sizes for toll-like receptor 2 and toll-like receptor 4 gene polymorphisms and interleukin-1β gene

Genes	Primers	Enzyme T°/time	Fragment (bp)	Ref.
TLR2	F: 5’-CACGGAGGCAGCGAGAAA-3’	-	286	[24]
del -196 to -174	R: 5’-CTGGGCCGTGCAAAGAAG-3’		ins/ins: 286
			ins/del: 286, 264
			del/del: 264
TLR4+896A/G	F: 5’-AGCATACTTAGACTACCACCTCGATG 3’	BstXI	131	[34]
rs4986790	R: 5’-GTTGCCATCCGAAATTATAAGAAAAG 3’	37 °C, 1 h	A/A: 131
			A/G: 131, 108
			G/G: 108
TLR4+1196C/T	F: 5’-GGTTGCTGTTCTCAAAGTGATTTTGGGAGAA-3’	HinfI	407	[33]
rs4986791	R: 5’-ACCTGAAGACTGGAGAGTGAGTTAAATGCT-3’	37 °C, 1 h	C/C: 407
			C/T: 407, 378
			T/T: 378
IL1-β	F: CATGTGACCTGCTCGTCAGT	HinfI	370	[47]
	R: CCCTAGGGATTGAGTCCACA	37 °C, 1 h	195, 175
TLR2	F: 5’-CACGGAGGCAGCGAGAAA-3’	BstXI	286	[24]
	R: 5’-CTGGGCCGTGCAAAGAAG-3’	37 °C, 1 h	188, 98

TLR: Toll-like receptor; IL: Interleukin; T^o: Temperature.

Table 2 Genotype and allele frequencies of toll-like receptor 2 (-196 to -174 del) and toll-like receptor 4 (rs4986790 and rs4986791) polymorphisms in gastric cancer, chronic gastritis and control groups

Genotypes/alleles	GC	C	CG
	n = 174 (%)	n = 225(%)	n = 208 (%)
TLR2 -196 to -174
ins/ins	116 (66.6)	189 (84.0)	160 (76.9)
del/ins	50 (28.7)	34 (15.1)	41 (19.7)
del/del	8 (4.7)	2 (0.9)	7 (3.4)
OR (95% CI)	2.62 (1.63-4.22) 1.57 (0.97-2.54)
P value	< 0.01 0.06
Alleles
ins	81.0	91.5	87.0
del	19.0	8.5	13.0
OR (95% CI)	2.53 (1.65-3.88) 1.65 (1.06-2.55)
P value	< 0.01 0.02
TLR4 +896A/G
(rs4986790)
AA	154 (88.5)	215 (95.5)	187 (89.9)
AG	20 (11.5)	10 (4.5)	21 (10.1)
GG	0	0	0
OR (95% CI)	2.79 (1.27-6.13) 2.41 (1.10-5.25)
P value	0.01 0.02
Alleles
A	94.0	97.0	95.0
G	6.0	3.0	5.0
OR (95% CI)	2.68 (1.23-5.81) 2.33 (1.08-5.02)
P value	0.01 0.02
TLR4 +1196C/T
(rs4986791)
CC	165 (94.8)	219 (97.3)	202 (97.1)
CT	9 (5.2)	6 (2.7)	6 (2.9)
TT	0	0	0
OR (95% CI)	1.99 (0.69-5.70) 1.08 (0.34-3.41)
P value	0.28 1.00
Alleles
C	98.0	99.0	98.0
T	2.0	1.0	2.0
OR (95% CI)	1.96 (0.69-5.57) 1.08 (0.34-3.38)
P value	0.29 1.00

OR: Odds ratio; GC: Gastric cancer; CG: Chronic gastritis; C: Control.

Table 3 Toll-like receptor 4 haplotype frequency distribution between gastric cancer, chronic gastritis and control groups

Haplotypes	GC (%)	C (%)	χ2	P value	CG (%)	C (%)	χ2	P value
TLR4 +896/+1196
A-C	91.4	95.7	6.802	< 0.01	94.0	95.7	1.361	0.24
G-C	63.0	31.0	5.247	0.02	46.0	31.0	1.598	0.20
A-T	21.0	11.0	1.461	0.22	NF	NF	NF	NF

Haplotype G-T not found. NF: Not found; GC: Gastric cancer; CG: Chronic gastritis; C: Control.

Table 4 Combined effect of toll-like receptor 2 (-196 to -174 del) and toll-like receptor 4 (rs4986790 and rs4986791) polymorphisms on risk of gastric cancer and chronic gastritis

Risk genotype	Groups
	GC (n = 174)	C (n = 225)	OR (95% CI)P value	CG (n = 208)	C (n = 225)	OR (95% CI)P value	GC (n =174)	CG (n = 225)	OR (95% CI)P value
Neither	113	187	1.00 (reference)	160	187	1.00 (reference)	113	160	1.00 (reference)
TLR2 ins/del or del/ del/TLR4+896 AG	10	4	4.13 (1.26-13.50)	11	4	3.21 (1.00-10.29)	10	11	1.28 (0.52-3.13)
			0.02			0.06			0.64
TLR2 ins/del or del/ del/TLR4+1196 CT	4	1	6.61 (0.73-59.99)	1	1	1.16 (0.07-18.84)	4	1	5.66 (0.62-51.37)
			0.07			1.00			0.16
TLR4+896 AG/ +1196 CT	1	1	1.65 (0.10-26.73)	3	1	3.50 (0.36-34.05)	1	3	0.47 (0.04-4.59)
			1.00			0.34			0.64

C: Control group; OR: Odds ratio; GC: Gastric cancer; CG: Chronic gastritis; TLR: Toll-like receptor.

Table 5 Distribution of risk factors, genotypes of toll-like receptor 2 (-196 to -174 del) and toll-like receptor 4 (rs4986790 and rs4986791), and odds ratios for gastric cancer, chronic gastritis and control groups

Variables	F (GC/C) %	OR (95% CI)	P value	F (CG/C) %	OR (95% CI)	P value
Gender
Female	23/49.7	Reference	< 0.01	51.0/50.3	Reference	0.96
Male	77/50.3	2.70 (1.66-4.41)		49.0/49.7	0.99 (0.66 -1.48)
Age (yr)
< 61	40.9/56.0	Reference	0.30	52.8/43.5	(< 53 yr) Reference	0.03
≥ 61	59.1/44.0	1.26 (0.81-1.98)		47.2/56.5	(≥ 53 yr) 0.64 (0.43-0.95)
Smoking
Nonsmokers	30.5/34.3	Reference	0.13	44.8/34.3	Reference	0.01
Smokers	69.5/65.7	0.67(0.39-1.13)		55.2/65.7	0.57 (0.37-0.87)
Alcohol
Nondrinkers	46.6/74.3	Reference	< 0.01	68.2/74.3	Reference	0.06
Drinkers	53.4/25.7	2.93 (1.76-4.87)		31.8/25.7	1.54 (0.97-2.44)
TLR2
ins/ins	66.6/84.0	Reference	< 0.01	76.9/84.0	Reference	0.18
ins/del	33.4/16.0	2.64 (1.56-4.44)		23.1/16.0	1.39 (0.84 -2.28)
del/del
TLR4 +896A/G (rs4986790)
AA	88.5/95.5	Reference	< 0.01	89.9/95.5	Reference	0.04
AG	11.5/4.5	3.19 (1.34-7.61)		10.1/4.5	2.29 (1.02-5.13)
TLR4 +1196C/T (rs4986791)
CC	94.8/97.3	Reference	0.63	97.1/97.3	Reference	0.91
CT	5.2/2.7	1.33 (0.41-4.33)		2.9/2.7	0.93 (0.28-3.05)

F: Frequency of individual (%); OR: Odds ratio; GC: Gastric cancer; CG: Chronic gastritis; C: Control; TLR: Toll-like receptor.