TLR Signaling Pathway Gene Polymorphisms, Gene–Gene and Gene–Environment Interactions in Allergic Rhinitis

Figures & data

Table 1 Gene Frequency Distribution of TLR2, TLR4 and CD14 Alleles

No.	SNPs	Gene	Location	Base Change	MAF			HWE P^b
					Database^a	Case	Control
1	rs7656411	TLR2	3ʹnear gene	G/T	0.419	0.482	0.451	0.168
2	rs76112010	TLR2	5ʹnear gene	A/G	0.136	0.203	0.169	0.661
3	rs7682814	TLR2	5ʹnear gene	A/G	0.167	0.202	0.172	0.286
4	rs10983755	TLR4	5’ near gene	A/G	0.267	0.127	0.154	0.457
5	rs11536889	TLR4	3ʹUTR	G/C	0.261	0.238	0.218	0.065
6	rs1927914	TLR4	5ʹnear gene	A/G	0.360	0.434	0.416	0.204
7	rs7873784	TLR4	3ʹUTR	G/C	0.122	0.274	0.259	0.537
8	rs2563298	CD14	3ʹUTR	A/C	0.151	0.123	0.184	0.067
9	rs2569190	CD14	5ʹUTR	A/G	0.500	0.380	0.412	0.988
10	rs2569191	CD14	5ʹnear gene	C/T	0.453	0.363	0.426	0.625

Notes: ^aMAF for CHB from the HapMap databases (http://www.hapmap.org) or PubMed (http://www.ncbi.nlm.nih.gov/snp). ^bTwo-sided χ² test for the allele frequencies of the controls.

Abbreviations: SNPs, single nucleotide polymorphisms; MAF, minor allele frequency; HWE, Hardy-Weinberg equilibrium.

Table 2 Distribution of Selected Variables Among Cases and Controls

Variables	Case (n = 452)		Control (n = 495)		P^a
	N	%	N	%
Age (years), mean ± SD	19.70 ± 12.60		20.10 ± 12.61		0.597
Gender
Male	302	66.8	303	61.2	0.073
Female	150	33.2	192	38.8
Duration of rhinitis (year), mean ± SD	6.62 ± 5.87
Total nasal symptoms (VAS), mean ± SD	5.25 ± 2.39
Serum total IgE (kU/L), median (IQR)^b	264.0 (120.6–593.5)		26.8 (11.1–52.2)		< 0.001*
Serum specific IgE (kUA/L), median (IQR)^b
Dermatophagoides pteronyssinus	29.0 (4.6–72.5)
Dermatophagoides farinae	24.1 (5.0–69.9)
Serum ECP (μg/L), median (IQR)^b	12.7 (5.2–28.7)		4.6 (3.1–7.5)		< 0.001*
With asthma^c
Yes	118	26.1
No	271	60.0

Notes: ^aDerived from two-sided χ² test for comparison of discrete variables and unpaired Student’s t-test for continuous variables. ^bSelective variables were transformed into logarithmic model before unpaired Student’s t-test between cases and controls. ^cSome information of concomitant asthma of allergic diseases was not available in cases. *Statistically significant (P < 0.05).

Abbreviations: IQR, interquartile range; ECP, eosinophil cationic protein.

Table 3 Genotype and Allele Frequencies in TLR2, TLR4 and CD14 Polymorphisms Among Cases and Controls

SNPs	Genotype	Case		Control		Crude OR (95% CI)	Adjusted OR (95% CI)^a	P^b
		N	%	N	%
TLR2
rs7656411		n = 441		n = 493
	GG	112	25.4	156	31.6	1.00 (reference)	1.00 (reference)	0.079
	GT	233	52.8	229	46.5	1.42 (1.05–1.92)	1.41 (1.04–1.91)
	TT	96	21.8	108	21.9	1.23 (0.86–1.79)	1.22 (0.84–1.76)
	GT/TT	329	74.6	337	68.4	1.36 (1.02–1.81)	1.35 (1.01–1.79)	0.035*
	T allele	0.482		0.451				0.186
rs76112010		n = 446		n = 485
	GG	287	64.4	331	68.3	1.00 (reference)	1.00 (reference)	0.144
	GA	137	30.7	141	29.1	1.12 (0.84–1.49)	1.15 (0.86–1.53)
	AA	22	4.96	13	2.7	1.95 (0.97–3.95)	1.98 (0.98–4.02)
	GA/AA	159	35.7	154	31.2	1.19 (0.91–1.53)	1.22 (0.93–1.60)	0.209
	A allele	0.203		0.172				0.089
rs7682814		n = 441		n = 486
	GG	285	64.6	330	67.9	1.00 (reference)	1.00 (reference)	0.074
	GA	134	30.4	145	29.8	1.07 (0.81–1.42)	1.09 (0.82–1.45)
	AA	22	5.00	11	2.26	2.32 (1.10–4.86)	2.29 (1.09–4.81)
	GA/AA	42	15.9	38	13.9	1.15 (0.88–1.52)	1.17 (0.90–1.55)	0.292
	A allele	0.202		0.172				0.097
TLR4
rs10983755		n = 423		n = 455
	GG	327	77.3	327	71.9	1.00 (reference)	1.00 (reference)	0.157
	GA	84	19.9	115	25.3	0.73 (0.53–1.01)	0.72 (0.52–0.98)
	AA	12	2.8	13	2.9	0.92 (0.42–2.05)	0.98 (0.44–2.18)
	GA/AA	96	22.7	128	28.1	0.75 (0.55–1.02)	0.74 (0.54–1.01)	0.065
	A allele	0.128		0.155				0.101
rs11536889		n = 427		n = 482
	GG	254	59.5	298	61.8	1.00 (reference)	1.00 (reference)	0.765
	GC	143	33.5	153	31.7	1.10 (0.83–1.46)	1.09 (0.82–1.45)
	CC	30	7.0	31	6.4	1.13 (0.67–1.93)	1.18 (0.69–2.01)
	GC/CC	173	40.5	184	38.1	1.10 (0.85–1.44)	1.10 (0.84–1.44)	0.471
	C allele	0.238		0.223				0.458
rs1927914		n = 411		n = 453
	AA	128	31.1	161	35.5	1.00 (reference)	1.00 (reference)	0.286
	AG	209	50.9	207	45.7	1.27 (0.94–1.72)	1.25 (0.92–1.69)
	GG	74	18.0	85	18.8	1.10 (0.74–1.62)	1.09 (0.73–1.63)
	AG/GG	283	68.9	292	64.5	1.22 (0.92–1.62)	1.20 (0.90–1.60)	0.171
	G allele	0.434		0.416				0.445
rs7873784		n = 433		n = 453
	GG	235	54.3	251	55.4	1.00 (reference)	1.00 (reference)	0.644
	GC	159	36.7	169	37.3	1.01 (0.76–1.33)	1.00 (0.76–1.33)
	CC	39	9.0	33	7.28	1.26 (0.77–2.07)	1.27 (0.77–2.01)
	GC/CC	198	45.7	202	44.6	1.05 (0.80–1.36)	1.05 (0.80–1.37)	0.734
	C allele	0.274		0.259				0.496
CD14
rs2563298		n = 434		n = 479
	CC	336	77.4	326	68.1	1.00 (reference)	1.00 (reference)	0.003*
	CA	89	20.5	131	27.3	0.66 (0.48–0.90)	0.65 (0.48–0.89)
	AA	9	2.1	22	4.60	0.40 (0.18–0.88)	0.40 (0.18–0.89)
	CA/AA	98	68.5	153	31.9	0.62(0.46–0.84)	0.62 (0.46–0.83)	0.002*
	A allele	0.123		0.183				<0.001*
rs2569190		n = 450		n = 481
	AA	174	38.7	166	34.5	1.00 (reference)	1.00 (reference)	0.351
	AG	210	46.7	233	48.4	0.86 (0.65–1.14)	0.86 (0.64–1.15)
	GG	66	14.7	82	17.1	0.78 (0.88–1.92)	0.76 (0.52–1.94)
	AG/GG	276	61.3	315	65.5	0.84 (0.52–1.31)	0.84 (0.64–1.09)	0.188
	G allele	0.380		0.413				0.150
rs2569191		n = 441		n = 472
	CC	187	42.4	153	32.4	1.00 (reference)	1.00 (reference)	0.008*
	CT	188	42.6	236	50.0	0.65 (0.49–0.87)	0.65 (0.48–0.87)
	TT	66	15.0	83	17.6	0.65 (0.44–0.96)	0.64 (0.44–0.95)
	CT/TT	254	57.6	319	67.6	0.65 (0.50–0.85)	0.65 (0.49–0.85)	0.002*
	T allele	0.363		0.426				0.006*

Notes: ^aAdjusted for age and sex in logistic regression model. ^bTwo-sided χ² test for the distributions of genotype and allele frequencies. *Statistically significant (P < 0.05).

Abbreviations: SNPs, single nucleotide polymorphisms; OR, odds ratio; CI, confidence interval.

Table 4 Stratification Analyses of TLR2, TLR4 and CD14 Polymorphisms in the Dominant Model in Cases and Controls

Variables	Subgroups	TLR2 rs7656411		P	CD14 rs2563298		P	CD14 rs2569191		P
		N (Case/Control)^a	Adjusted OR (95% CI)^b		N (Case/Control)^a	Adjusted OR (95% CI)^b		N (Case/Control)^a	Adjusted OR (95% CI)^b
Age (years)	< 18	247/239	1.33 (0.88–1.99)	0.173	244/239	0.54 (0.36–0.80)	0.003**	247/236	0.65 (0.44–0.94)	0.022*
	≥ 18	194/254	1.36 (0.91–2.06)	0.163	191/240	0.73 (0.47–1.13)	0.164	194/236	0.66 (0.44–0.98)	0.037*
Gender	Male	295/303	1.60 (1.12–2.30)	0.011*	289/296	0.75 (0.52–1.07)	0.115	293/289	0.66 (0.47–0.93)	0.018*
	Female	146/190	0.97 (0.60–1.57)	0.894	146/183	0.42 (0.25–0.72)	0.002**	148/183	0.62 (0.40–0.98)	0.038*
Asthma	No	263/493	1.28 (0.81–2.03)	0.292	261/479	0.45 (0.27–0.75)	0.002**	262/472	0.53 (0.35–0.81)	0.003**
	Yes	116/493	1.61 (1.13–2.28)	0.007*	115/479	0.60 (0.42–0.85)	0.006*	116/472	0.70 (0.51–0.96)	0.027*
VAS	< 5	247/493	1.25 (0.88–1.77)	0.207	241/479	0.65 (0.45–0.92)	0.017*	246/472	0.72 (0.52–0.99)	0.048*
	≥ 5	194/493	1.45 (0.99–2.12)	0.055	193/479	0.60 (0.40–0.88)	0.013*	195/472	0.57 (0.41–0.80)	0.001**
Total IgE^c	Lower	135/493	1.41 (0.99–2.00)	0.059	234/479	0.45 (0.31–0.67)	<0.0001**	237/472	0.61 (0.44–0.84)	0.002**
	Higher	206/493	1.31 (0.90–1.89)	0.154	200/479	0.82 (0.57–1.19)	0.339	204/472	0.71 (0.50–0.99)	0.047*

Notes: ^aControls were stratified accordingly when dividing cases into age and gender subgroups, while they were kept as a whole in the situation of the other subgroups. ^bAdjusted for age and gender in logistic regression model. ^cLower: below the 90th percentile of logarithmic total IgE level; Higher: above the 90th percentile of logarithmic total IgE level. *Nominally significant (P < 0.05) but did not withstand Bonferroni correction. **Significant after Bonferroni correction. Dominant model: MW/MM vs WW; MW: heterozygotes; MM: mutation homozygotes; WW: wild homozygotes.

Abbreviations: OR, odds ratio; CI, confidence interval; VAS, visual analogue scale.

Table 5 Multifactor Dimensionality Reduction Models for Locus–Locus Interactions

Model^a	CV Training	CV Testing	CV Consistency	OR (95% CI)	P
A10	0.5544	0.5352	8/10	1.5555 (1.1468–2.1099)	0.0036*
A3, A10	0.5759	0.5298	6/10	1.8436 (1.3591–2.5009)	< 0.0001*
A1, A6, A8	0.6074	0.5308	6/10	2.4043 (1.7642–3.2768)	< 0.0001*

Notes: ^aA1: rs7656411; A3; rs7682814; A6: rs1927914; A8: rs2563298; A10: rs2569191. *Statistically significant (P < 0.05).

Abbreviations: CV, cross-validation; OR, odds ratio; CI, confidence interval.

Figure 1 Tree diagram of the best genotype models. The distance between single nucleotide polymorphisms (SNPs) indicates the intensity of the interactions. The color indicates the type of interactions. Red, orange, and green denote strong, moderate and weak interactions, respectively. A1: rs7656411; A3: rs7682814; A6: rs1927914; A8: rs2563298; A10: rs2569191.

Table 6 Statistics of Environmental Factors in AR Case Group

Variables	N	%
Smoke
No	212	89.5
Yes	25	10.5
Living or working floors
< 4 floor	125	59.2
≥ 4 floor	86	40.8
Daytime sunshine
< 4 hours	30	13.4
≥ 4 hours	194	86.6
Recently renovated or purchased furniture
No	110	46.8
Yes	125	53.2
Polluting enterprises nearby
No	176	81.5
Yes	40	18.5
Distance to main road
< 1000 meters	170	78.3
≥ 1000 meters	47	21.7
Time of using computer per day
< 3 hours	143	72.2
≥ 3 hours	55	27.8
Cooking fumes
No	125	54.3
Yes	105	45.7
Pet-keeping
No	219	93.6
Yes	15	6.4

Abbreviation: AR, allergic rhinitis.

Table 7 Association Between Polymorphisms and Environmental Factors in the Dominant Model Among AR Case Group

SNPs	Genotype	Floors		Adjusted OR (95% CI)^a	P	Sunlight Hours		Adjusted OR (95% CI)^a	P	Pet-Keeping		Adjusted OR (95% CI)^a	P
		< 4F	≥ 4F			< 4h	≥ 4h			No	Yes
TLR4 rs10983755	GG	111	62	1.00 (reference)	0.048*	20	131	1.00 (reference)	0.730	147	11	1.00 (reference)	0.998
	GA/AA	41	17	0.51 (0.27–0.99)		7	51	1.18 (0.47–2.99)		57	4	1.00 (0.30–3.33)
TLR4 rs11536889	GG	76	49	1.00 (reference)	0.515	16	121	1.00 (reference)	0.268	128	14	1.00 (reference)	0.043*
	GC/CC	44	34	1.21 (0.68–2.15)		14	65	0.64 (0.29–1.41)		82	1	0.12 (0.02–0.93)
TLR4 rs7873784	GG	67	48	1.00 (reference)	0.725	22	95	1.00 (reference)	0.027*	118	5	1.00 (reference)	0.128
	GC/CC	55	36	0.90 (0.52–1.59)		8	94	2.66 (1.12–6.34)		96	10	2.38 (0.78–7.27)
CD14 rs2569190	AA	39	33	1.00 (reference)	0.352	15	64	1.00 (reference)	0.049*	79	5	1.00 (reference)	0.812
	AG/GG	85	53	0.76 (0.42–1.36)		15	129	2.22 (1.00–4.90)		139	10	1.15 (0.37–3.52)
CD14 rs2569191	CC	45	39	1.00 (reference)	0.186	18	76	1.00 (reference)	0.022*	94	6	1.00 (reference)	0.777
	CT/TT	79	46	0.68 (0.39–1.20)		12	116	2.56 (1.15–5.73)		123	9	1.17 (0.40–3.44)

Notes: ^aAdjusted for age and gender in logistic regression model. *Nominally significant (P < 0.05) but did not withstand Bonferroni correction. Dominant model: MW/MM vs WW; MW: heterozygotes; MM: mutation homozygotes; WW: wild homozygotes.

Abbreviations: AR, allergic rhinitis; SNPs, single nucleotide polymorphisms; OR, odds ratio; CI, confidence interval.

Table 8 Functional Annotation Information of 10 SNPs

SNPs	Promoter Histone Marks	Enhancer Histone Marks	DNAse	Proteins Bound	eQTL Results	Motifs Changed	MicroRNA^a	Function Annotation	Score^b
rs7656411	BLD	BLD			12 eQTL results	PU.1, Rad21			5
rs76112010		BLD							4
rs7682814						4 altered motifs			6
rs10983755			BLD		2 eQTL results	AIRE, DMRT4, Tgif1			7
rs11536889		12 tissues	4 tissues	STAT3	4 eQTL results	Ik-1	hsa-miR-1208, hsa-miR-1236	3ʹUTR	4
rs1927914		BLD			4 eQTL results	6 altered motifs			6
rs7873784		BLD			3 eQTL results		hsa-let-7d-3p, hsa-miR-16-1-3p, hsa-miR-4528	3ʹUTR	5
rs2563298	13 tissues	7 tissues			41 eQTL results	Myb, SIX5	hsa-miR-451a	3ʹUTR	6
rs2569190	20 tissues	11 tissues	25 tissues	8 bound proteins	49 eQTL results	5 altered motifs		5ʹUTR	1b
rs2569191	BLD, LIV	4 tissues			2 eQTL results	Mef2, Pou2f2, TATA			5

Notes: ^aPredicted possible binding microRNA by SNPinfo and MirSNP. ^bBased on RegulomeDB.

Abbreviations: SNPs, single nucleotide polymorphisms; BLD, blood; LIV, liver; eQTL, expression quantitative trait locus; UTR, untranslated region.

Figure 2 In silico prediction of CD14 folding structures and minimum free energy (MFE) changes corresponding to rs2563298 A to C allele. The arrows indicated the changes in structure caused by rs2563298.