Association between TLR-9 polymorphisms and colon cancer susceptibility in Saudi Arabian female patients

Figures & data

Table 1 Clinical characteristics of the subjects selected for genotyping

Characteristics	Cancer (n=115)	Control (n=102)
Gender
Male	66	60
Female	49	42
Age (years)
Male	55.50±13.94	53.24±17.4
Female	56.95±13.70	50.78±15.21
Localization
Colon	76	–
Rectum	39	–
Therapy
Chemotherapy
Yes	3	–
No	112	–
Radiotherapy
Yes	5	–
No	110	–

Figure 1 TLR-9 mRNA expression in colon cancer cells and colon cancer tissues.

Notes: Total RNA of tissues was extracted from matching normal and colon cancer tissues, reverse-transcribed into cDNA, and then used to measure TLR-9 mRNA expression with specific primers. TLR-9 expression in colon cancer tissues and matching control tissues is shown as mean ± SD.
Abbreviations: cDNA, complementary DNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Table 2 Genotype frequencies of TLR-9 gene polymorphisms in colorectal cancer and controls

Gene	SNP ID	Genotype	Case, n (%)	Control, n (%)	OR	95% CI	χ^2 value	P-value
TLR-9	rs187084	CC	47 (0.43)	34 (0.35)	Ref
		CT	47 (0.43)	53 (0.53)	0.642	0.355–1.158	2.18	0.13991
		TT	15 (0.14)	12 (0.12)	0.904	0.376–2.176	0.05	0.82221
		CT + TT	62 (0.57)	65 (0.65)	0.690	0.393–1.210	1.68	0.19487
		C	141 (0.65)	121 (0.61)	Ref
		T	77 (0.35)	77 (0.39)	0.858	0.576–1.278	0.57	0.45166
	rs352139	AA	33 (0.29)	31 (0.30)	Ref
		AG	55 (0.49)	55 (0.54)	0.939	0.507–1.740	0.04	0.84243
		GG	26 (0.23)	16 (0.16)	1.527	0.691–3.372	1.10	0.29448
		AG + GG	81 (0.71)	71 (0.70)	1.072	0.597–1.923	0.05	0.81642
		A	121 (0.54)	117 (0.57)	Ref
		G	107 (0.48)	87 (0.43)	1.189	0.813–1.740	0.80	0.37163
	rs352144	AA	2 (0.02)	0 (0)	Ref
		AC	5 (0.04)	7 (0.07)	0.147	0.006–3.706	2.33	0.12663
		CC	110 (0.94)	94 (0.93)	0.234	0.011–4.932	1.70	0.19294
		AC + CC	115 (0.98)	101 (1)	0.228	0.011–4.796	1.74	0.18682
		A	9 (0.04)	7 (0.03)	Ref
		C	225 (0.96)	195 (0.97)	0.897	0.328–2.455	0.04	0.83298

Abbreviations: CI, confidence interval; OR, odds ratio; Ref, reference; SNP, single-nucleotide polymorphism.

Figure 2 LD of the studied TLR-9 loci rs187084, rs352139, and rs352144 in colorectal cancer case and control groups.

Abbreviation: LD, linkage disequilibrium.

Table 3A Genotype frequencies of TLR-9 gene polymorphism in patients younger than 57 years with colorectal cancer

Gene	SNP ID	Genotype	Case, n (Frequency in %)	Control, n (Frequency in %)	OR	95% CI	χ^2 value	P-value
TLR-9	rs187084	CC	22 (0.42)	16 (0.27)	Ref
		CT	23 (0.43)	35 (0.58)	0.478	0.208–1.098	3.07	0.07989
		TT	8 (0.15)	9 (0.15)	0.646	0.205–2.041	0.56	0.45578
		CT + TT	31 (0.58)	44 (0.85)	0.512	0.232–1.130	2.78	0.09558
		C	67 (0.63)	67 (0.56)	Ref
		T	39 (0.37)	53 (0.44)	0.736	0.431–1.256	1.27	0.26013
	rs352139	AA	20 (0.36)	24 (0.39)	Ref
		AG	27 (0.48)	28 (0.45)	1.157	0.523–2.562	0.13	0.71882
		GG	9 (0.16)	10 (0.16)	1.080	0.367–3.175	0.02	0.88876
		AG + GG	36 (0.64)	38 (0.61)	1.137	0.538–2.403	0.11	0.73687
		A	67 (0.60)	76 (0.61)	Ref
		G	45 (0.40)	48 (0.39)	1.063	0.630–1.794	0.05	0.81763
	rs352144	AA	2 (0.03)	0 (0)	Ref
		AC	4 (0.07)	4 (0.06)	0.200	0.007–5.453	1.67	0.19671
		CC	53 (0.90)	58 (0.94)	0.183	0.009–3.897	2.15	0.14284
		AC + CC	57 (0.97)	62 (1)	0.184	0.009–3.914	2.14	0.14378
		A	8 (0.07)	4 (0.03)	Ref
		C	110 (0.93)	120 (0.97)	0.458	0.134–1.565	1.62	0.20304

Abbreviations: CI, confidence interval; OR, odds ratio; Ref, reference; SNP, single-nucleotide polymorphism.

Table 3B Genotype frequencies of TLR-9 gene polymorphism in patients older than 57 years with colorectal cancer

Gene	SNP ID	Genotype	Case, n (Frequency in %)	Control, n (Frequency in %)	OR	95% CI	χ^2 value	P-value
TLR-9	rs187084	CC	25 (0.45)	18 (0.46)	Ref
		CT	24 (0.43)	18 (0.46)	0.960	0.406–2.270	0.01	0.92592
		TT	7 (0.12)	3 (0.08)	1.680	0.382–7.395	0.48	0.48975
		CT + TT	31 (0.55)	21 (0.54)	1.063	0.468–2.415	0.02	0.88428
		C	74 (0.66)	54 (0.69)	Ref
		T	38 (0.34)	24 (0.31)	1.155	0.622–2.147	0.21	0.64774
	rs352139	AA	13 (0.22)	7 (0.17)	Ref
		AG	28 (0.48)	27 (0.68)	0.558	0.193–1.612	1.18	0.27836
		GG	17 (0.30)	6 (0.15)	1.526	0.413–5.642	0.40	0.52559
		AG + GG	45 (0.78)	33 (0.83)	0.734	0.264–2.042	0.35	0.55304
		A	54 (0.47)	41 (0.51)	Ref
		G	62 (0.53)	39 (0.49)	1.207	0.682–2.135	0.42	0.51772
	rs352144	AA	0 (0)	0 (0)	Ref
		AC	1 (0.02)	3 (0.08)	0.429	0.005–33.596	–	1.000
		CC	57 (0.98)	36 (0.92)	1.575	0.031–81.149	–	1.000
		AC + CC	58 (1)	39 (1)	1.481	0.029–76.201	–	1.000
		A	1 (0.01)	3 (0.04)	Ref
		C	115 (0.99)	75 (0.96)	4.600	0.470–45.053	2.06	0.30785

Abbreviations: CI, confidence interval; OR, odds ratio; Ref, reference; SNP, single-nucleotide polymorphism.

Table 4A Genotype frequencies of TLR-9 gene polymorphism in male patients with colorectal cancer

Gene	SNP ID	Genotype	Case, n (Frequency in %)	Control, n (Frequency in %)	OR	95% CI	χ^2 value	P-value
TLR-9	rs187084	CC	22 (0.35)	19 (0.33)	Ref
		CT	30 (0.48)	35 (0.62)	0.740	0.338–1.621	0.57	0.45162
		TT	11 (0.17)	3 (0.05)	3.167	0.768–13.05	2.70	0.10042
		CT + TT	41 (0.65)	38 (0.67)	0.932	0.438–1.985	0.03	0.85473
		C	74 (0.59)	73 (0.64)	Ref
		T	52 (0.41)	41 (0.36)	1.251	0.743–2.108	0.71	0.39956
	rs352139	AA	19 (0.30)	15 (0.25)	Ref
		AG	31 (0.48)	34 (0.58)	0.720	0.313–1.657	0.60	0.43896
		GG	14 (0.22)	10 (0.17)	1.105	0.384–3.180	0.03	0.85273
		AG + GG	45 (0.70)	44 (0.75)	0.807	0.365–1.787	0.28	0.59734
		A	69 (0.54)	64 (0.54)	Ref
		G	59 (0.46)	54 (0.46)	1.013	0.613–1.674	0	0.95849
	rs352144	AA	2 (0.03)	0 (0)	Ref
		AC	1 (0.01)	3 (0.05)	0.086	0.002–3.101	3.00	0.08326
		CC	64 (0.96)	55 (0.95)	0.232	0.011–4.945	1.69	0.19299
		AC + CC	65 (0.97)	58 (1)	0.224	0.011–4.760	1.76	0.18469
		A	5 (0.04)	3 (0.03)	Ref
		C	129 (0.96)	113 (0.97)	0.685	0.160–2.930	0.26	0.72813

Abbreviations: CI, confidence interval; OR, odds ratio; Ref, reference; SNP, single-nucleotide polymorphism.

Table 4B Genotype frequencies of TLR-9 gene polymorphism in female patients with colorectal cancer

Gene	SNP ID	Genotype	Case, n (Frequency in %)	Control, n (Frequency in %)	OR	95% CI	χ^2 value	P-value
TLR-9	rs187084	CC	25 (0.54)	15 (0.37)	Ref
		CT	17 (0.37)	18 (0.44)	0.567	0.225–1.424	1.47	0.22539
		TT	4 (0.09)	8 (0.19)	0.300	0.077–1.169	3.18	0.07439
		CT + TT	21 (0.46)	26 (0.63)	0.485	0.205–1.146	2.75	0.09703
		C	67 (0.73)	48 (0.59)	Ref
		T	25 (0.27)	34 (0.41)	0.527	0.279–0.995	3.95	0.04686
	rs352139	AA	14 (0.28)	15 (0.36)	Ref
		AG	24 (0.48)	21 (0.50)	1.224	0.481–3.118	0.18	0.67089
		GG	12 (0.24)	6 (0.14)	2.143	0.632–7.266	1.52	0.21765
		AG + GG	36 (0.72)	27 (0.64)	1.429	0.591–3.454	0.63	0.42761
		A	52 (0.52)	51 (0.61)	Ref
		G	48 (0.48)	33 (0.39)	1.427	0.792–2.568	1.41	0.23559
	rs352144	AA	0 (0)	0 (0)	Ref
		AC	4 (0.08)	4 (0.1)	1.000	0.016–62.3	–	1.000
		CC	46 (0.92)	38 (0.9)	1.208	0.023–62.29	–	1.000
		AC + CC	50 (1)	42 (1)	1.188	0.023–61.16	–	1.000
		A	4 (0.04)	4 (0.05)	Ref
		C	96 (0.96)	80 (0.95)	1.200	0.291–4.951	0.06	1.000

Note: Bold values are statistically significantly different.

Abbreviations: CI, confidence interval; OR, odds ratio; Ref, reference; SNP, single-nucleotide polymorphism.

Table 5A Genotype frequencies of TLR-9 gene polymorphisms in colorectal tumors located in the colon area

Gene	SNP ID	Genotype	Case, n (Frequency in %)	Control, n (Frequency in %)	OR	95% CI	χ^2 value	P-value
TLR-9	rs187084	CC	30 (0.45)	34 (0.35)	Ref
		CT	27 (0.41)	53 (0.53)	0.577	0.294–1.134	2.56	0.10952
		TT	9 (0.14)	12 (0.12)	0.850	0.315–2.297	0.10	0.74850
		CT + TT	36 (0.55)	65 (0.65)	0.628	0.332–1.188	2.06	0.15130
		C	87 (0.66)	121 (0.61)	Ref
		T	45 (0.34)	77 (0.39)	0.813	0.513–1.287	0.78	0.37640
	rs352139	AA	23 (0.34)	31 (0.30)	Ref
		AG	32 (0.47)	55 (0.54)	0.784	0.392–1.569	0.47	0.49166
		GG	13 (0.19)	16 (0.16)	1.095	0.441–2.718	0.04	0.84470
		AG + GG	45 (0.66)	71 (0.70)	0.854	0.443–1.646	0.22	0.63780
		A	78 (0.57)	117 (0.57)	Ref
		G	58 (0.43)	87 (0.43)	1.000	0.645–1.551	0	1.000
	rs352144	AA	0 (0)	0 (0)	Ref
		AC	4 (0.06)	7 (0.07)	0.600	0.01–35.86	0.001	1.000
		CC	65 (0.94)	94 (0.93)	0.693	0.014–35.37	0.001	1.000
		AC + CC	69 (1)	101 (1)	0.685	0.013–34.92	0	1.000
		A	4 (0.03)	7 (0.03)	Ref
		C	134 (0.97)	195 (0.97)	1.203	0.345–4.189	0.08	1.000

Abbreviations: CI, confidence interval; OR, odds ratio; Ref, reference; SNP, single-nucleotide polymorphism.

Table 5B Genotype frequencies of TLR-9 gene polymorphisms in colorectal cancer tumors located in rectal area

Gene	SNP ID	Genotype	Case, n (Frequency in %)	Control, n (Frequency in %)	OR	95% CI	χ^2 value	P-value
TLR-9	rs187084	CC	14 (0.42)	34 (0.35)	Ref
		CT	15 (0.46)	53 (0.53)	0.687	0.295–1.602	0.76	0.38390
		TT	4 (0.12)	12 (0.12)	0.810	0.223–2.945	0.10	0.74819
		CT + TT	19 (0.58)	65 (0.65)	0.710	0.317–1.588	0.70	0.40332
		C	43 (0.65)	121 (0.61)	Ref
		T	23 (0.35)	77 (0.39)	0.841	0.470–1.503	0.34	0.55786
	rs352139	AA	6 (0.18)	31 (0.30)	Ref
		AG	17 (0.50)	55 (0.54)	1.597	0.570–4.471	0.80	0.37026
		GG	11 (0.32)	16 (0.16)	3.552	1.11–11.37	4.81	0.02825
		AG + GG	28 (0.82)	71 (0.70)	2.038	0.767–5.415	2.09	0.14812
		A	29 (0.43)	117 (0.57)	Ref
		G	39 (0.57)	87 (0.43)	1.809	1.038–3.150	4.44	0.03519
	rs352144	AA	1 (0.03)	0 (0)	Ref
		AC	1 (0.03)	7 (0.07)	0.067	0.002–2.563	3.94	0.04722
		CC	33 (0.94)	94 (0.93)	0.118	0.005–2.972	2.79	0.09506
		AC + CC	34 (0.97)	101 (1)	0.113	0.005–2.847	2.91	0.08819
		A	3 (0.04)	7 (0.03)	Ref
		C	67 (0.96)	195 (0.97)	0.802	0.202–3.189	0.10	0.73581

Note: Bold values are statistically significantly different.

Abbreviations: CI, confidence interval; OR, odds ratio; Ref, reference; SNP, single-nucleotide polymorphism.

Table S1 Characteristics of selected polymorphisms in TLR-9

SNP ID	Chromosome/position	Nucleotide change	Position	Region	Minor allele frequency (%)
					Cases	Controls
rs187084	3/52227015	C>T	4149	Promoter	77 (0.35)	77 (0.39)
rs352139	3/52216841	A>G	6808	Intron	107 (0.48)	87 (0.43)
rs352144	3/52219937	A>C	3712	Upstream variant	225 (0.96)	195 (0.97)

Abbreviation: SNP, single-nucleotide polymorphism.