GARP Polymorphisms Associated with Susceptibility to House Dust Mite-Sensitized Persistent Allergic Rhinitis in a Chinese Population

Figures & data

Table 1 Primers and Probes for Genotyping by TaqMan Assay

NCBI rs No.	Base Change	Primers^a	Probes^a
rs947998	G>T	F: GCCTGATCTTTGAAAACACTACACA	G allele: FAM-CCCAGGCCGCAGC-MGB
		R: CCACACTGCTTCTCCAAAATTAGTT	T allele: HEX-CCCAGGCCTCAGC-MGB
rs79525962	C>T	F: GGTTCCCCTGCAGGTTGAG	G allele: FAM-ACACCTTTGCCAATC-MGB
		R: CTTAAGCCACAATGCCCTGGAG	A allele: HEX-ATACACCTTTACCAATCT-MGB
rs1320646	G>A	F: TCTTCTGAGAATGACTTTCAGTCTCTCT	G allele: FAM-TAGGACCGGAAGAGA-MGB
		R: CCAGATCCGAGACACACTCGTA	A allele: HEX-TTAGGACCAGAAGAGAG-MGB
rs3781699	A>C	F: GCCAAGCTGGGTGCAAAA	T allele: FAM-AGAACCAGATATCTAAG-MGB
		R: GCTGCTGAGCCAGGAGCTAA	G allele: HEX-AGAACCAGATAGCTAAGGT-MGB
rs1803627	T>G	F: TCTCTCTGTGCTCTTGCATTCTCT	T allele: FAM-ATTCCCTTTTCCTCTATTGA-MGB
		R: TCATTCTCTTCCTAAGCCTCAGTTTC	G allele: HEX-TTTTCCTCTATGGAGCAGA-MGB
rs7685	T>G	F: GCCTGATCTTTGAAAACACTACACA	G allele: FAM-CCCAGGCCGCAGC-MGB
		R: CCACACTGCTTCTCCAAAATTAGTT	T allele: HEX-CCCAGGCCTCAGC-MGB

Note: ^aThe alleles were arrayed as the location of the primers or probes from 5’ to 3’.

Table 2 Distribution of Selected Variables Among Cases and Controls

Variables	Case (n=534)		Control (n=451)		P
	N	%	N	%
Age (years), median (quartiles)	16.0 (10.0–27.0)		16.0 (10.0–29.0)		0.535^a
Gender
Male	356	66.7	278	61.6	0.101^a
Female	178	33.3	173	38.4
Serum total IgE (kU/L), median (quartiles)	255.1 (118.9–560.5)		25.2 (10.4–47.2)		< 0.001^b
Specific IgE (kUA/L), median (quartiles)
Der p	27.4 (6.1–69.5)
Der f	23.1 (6.0–63.0)
Rhinitis severity (VAS score)^c
Mild (VAS score ≤ 5)	208	45.6
Moderate-to-severe (VAS score > 5)	248	54.4
Concomitant asthma^c
Yes	124	27.5
No	327	72.5
Family history of allergic rhinitis^c
Yes	154	34.4
No	294	65.6

Notes: ^aTwo-sided χ² test for comparison of discrete variables and nonparametric test for continuous variables with non-normal distribution. ^bSelective variables were transformed into logarithmic model before unpaired Student’s t-test between cases and controls. ^cData about VAS score, concomitant asthma, and family history of allergic diseases were not available in some cases.

Abbreviations: Der p, Dermatophagoides pteronyssinus; Der f, Dermatophagoides farina; VAS, visual analogue scales.

Table 3 Primary Information of Six SNPs in the GARP Gene

SNP No.	NCBI rs No.	Chromosome Position^a	Location	Base Change	MAF			P for HWE^c	Genotyped (%)
					Database^b	Case	Control
1	rs947998	76672762	5’ near gene	G>T	0.305	0.310	0.317	0.290	98.1
2	rs79525962	76660374	exon	C>T	0.092	0.129	0.095	0.274	99.2
3	rs1320646	76659003	3ʹUTR	G>A	0.111	0.093	0.109	0.893	99.3
4	rs3781699	76658741	3ʹUTR	A>C	0.407	0.326	0.362	0.392	99.8
5	rs1803627	76658300	3ʹUTR	T>G	0.061	0.087	0.081	0.051	99.2
6	rs7685	76657595	3ʹUTR	T>G	0.350	0.332	0.355	0.548	99.2

Notes: ^aSNP position in NCBI dbSNP (http://www.ncbi.nlm.nih.gov/snp). ^bMAF for CHB from the HapMap databases (http://hapmap.ncbi.nlm.nih.gov) or NCBI dbSNP (http://www.ncbi.nlm.nih.gov/snp). ^cHWE P-value in the control group using a goodness-of-fit χ² test.

Abbreviations: SNP, single nucleotide polymorphism; GARP, glycoprotein A repetitions predominant; MAF, minor allele frequencies; HWE, Hardy-Weinberg equilibrium; UTR, untranslated region; CHB, Han Chinese in Beijing, China.

Table 4 Genotypes and Allele Frequencies in GARP Polymorphisms Among Cases and Controls

SNP No.	NCBI rs No.	Genotypes	Case		Control		Crude OR (95% CI)	Adjusted OR (95% CI)^a	P^b
			N	%	N	%
1	rs947998		n=531		n=435
		GG	249	46.9	198	45.5	1.000 (reference)	1.000 (reference)	0.911
		GT	235	44.3	198	45.5	0.944 (0.724–1.231)	0.937 (0.718–1.225)
		TT	47	8.9	39	9.0	0.958 (0.603–1.524)	0.988 (0.620–1.574)
		GT+TT	282	53.1	237	54.5	0.946 (0.734–1.220)	0.946 (0.733–1.221)	0.670
		T allele	0.310		0.317				0.725
2	rs79525962		n=533		n=444
		CC	406	76.2	362	81.5	1.000 (reference)	1.000 (reference)	0.034
		CT	117	22.0	80	18.0	1.304 (0.949–1.792)	1.319 (0.959–1.814)
		TT	10	1.9	2	0.5	4.458 (0.970–20.481)	4.225 (0.918–19.452)
		CT+TT	127	23.8	82	18.5	1.381 (1.011–1.886)	1.393 (1.019–1.904)	0.042
		T allele	0.129		0.095				0.018
3	rs1320646		n=532		n=446
		GG	442	83.1	354	79.4	1.000 (reference)	1.000 (reference)	0.169
		GA	81	15.2	87	19.5	0.746 (0.534–1.041)	0.742 (0.531–1.037)
		AA	9	1.7	5	1.1	1.442 (0.479–4.340)	1.460 (0.484–4.405)
		GA+AA	90	16.9	92	20.6	0.783 (0.568–1.082)	0.781 (0.565–1.079)	0.138
		A allele	0.093		0.109				0.250
4	rs3781699		n=534		n=449
		AA	240	44.9	187	41.6	1.000 (reference)	1.000 (reference)	0.151
		AC	240	44.9	199	44.3	0.940 (0.719–1.229)	0.932 (0.713–1.220)
		CC	54	10.1	63	14.0	0.668 (0.443–1.007)	0.646 (0.427–0.976)
		AC+CC	294	55.1	262	58.4	0.874 (0.679–1.127)	0.863 (0.670–1.114)	0.299
		C allele	0.326		0.362				0.093
5	rs1803627		n=534		n=443
		TT	446	83.5	377	85.1	1.000 (reference)	1.000 (reference)	0.574
		TG	83	15.5	60	13.5	1.169 (0.816–1.675)	1.171 (0.817–1.678)
		GG	5	0.9	6	1.4	0.704 (0.213–2.326)	0.701 (0.212–2.320)
		TG+GG	88	16.5	66	14.9	1.127 (0.796–1.595)	1.128 (0.797–1.597)	0.500
		G allele	0.087		0.081				0.645
6	rs7685		n=532		n=445
		TT	237	44.5	188	42.2	1.000 (reference)	1.000 (reference)	0.492
		TG	237	44.5	198	44.5	0.949 (0.726–1.242)	0.942 (0.719–1.233)
		GG	58	10.9	59	13.3	0.780 (0.518–1.175)	0.757 (0.501–1.143)
		TG+GG	295	55.5	257	57.8	0.911 (0.706–1.174)	0.899 (0.697–1.161)	0.470
		G allele	0.332		0.355				0.281

Notes: ^aAdjusted for age and gender in multivariate logistic regression model. Bold values denote statistical significance at the P < 0.05 level. ^bTwo-sided χ² test for the distributions of genotype and allele frequencies.

Abbreviations: GARP, glycoprotein A repetitions predominant; SNP, single nucleotide polymorphism; OR, odds ratio; CI, confidence interval.

Table 5 Stratification Analysis of rs79525962 in the Dominant Model in PER Subgroups

Variables	Subcategory	Case	Control	Adjusted OR (95% CI)^a
Age (years)		n=533	n=444
	< 16	257	225	1.076 (0.693–1.671)
	≥ 16	276	219	1.745 (1.103–2.760)
Gender		n=533	n=444
	Male	356	273	1.241 (0.836–1.840)
	Female	177	171	1.708 (1.021–2.856)
Concomitant asthma		n=450
	No	326		1.000 (reference)
	Yes	124		0.936 (0.565–1.552)
Family history of allergic diseases		n=447
	No	293		1.000 (reference)
	Yes	154		0.987 (0.620–1.571)
Rhinitis severity (VAS score)		n=455
	VAS score ≤ 5	207		1.000 (reference)
	VAS score > 5	248		0.909 (0.585–1.411)
Serum total IgE^b		n=529	n=442
	Lower level	431	442	1.000 (reference)
	Higher level	98		1.144 (0.696–1.880)

Notes: ^aAdjusted for age and gender in multivariate logistic regression model. Bold values denote statistical significance at the P < 0.05 level. ^bLower level: below the 90th percentile of logarithmic total IgE; higher level: above the 90th percentile of logarithmic total IgE.

Abbreviations: Dominant model: MW+MM/WW; MW: heterozygotes; MM: mutation homozygotes; WW: wild homozygotes. PER, persistent allergic rhinitis; VAS, visual analogue scale; OR, odds ratio; CI, confidence interval.

Table 6 Genotype Frequencies of GARP Polymorphisms in the Dominant Model in PER Patients with and without Asthma

SNP No.	NCBI rs No.	Case with Asthma vs Control	Case without Asthma vs Control
		Adjusted OR (95% CI)^a	Adjusted OR (95% CI)^a
1	rs947998	0.670 (0.445–1.008)	1.140 (0.853–1.525)
2	rs79525962	1.249 (0.765–2.041)	1.410 (0.993–2.003)
3	rs1320646	0.948 (0.570–1.576)	0.714 (0.490–1.041)
4	rs3781699	0.919 (0.612–1.380)	0.951 (0.712–1.271)
5	rs1803627	0.950 (0.536–1.684)	1.199 (0.813–1.768)
6	rs7685	1.060 (0.703–1.598)	0.945 (0.707–1.263)

Note: ^aAdjusted for age and gender in multivariate logistic regression model.

Abbreviations: Dominant model, MW+MM/WW; MW, heterozygotes; MM, mutation homozygotes; WW, wild homozygotes; GARP, glycoprotein A repetitions predominant; PER, persistent allergic rhinitis; SNP, single nucleotide polymorphism; OR, odds ratio; CI, confidence interval.

Table 7 Associations Between GARP Polymorphisms and AR-Related Phenotypes

SNP No.	NCBI rs No.	P for Serum Total IgE^a		P for Specific IgE^b		P for VAS Score^c
		Case	Control	Der p	Der f
1	rs947998	0.782	0.780	0.179	0.213	0.139
2	rs79525962	0.608	0.270	0.820	0.800	0.524
3	rs1320646	0.713	0.442	0.543	0.244	0.518
4	rs3781699	0.173	0.938	0.803	0.434	0.736
5	rs1803627	0.944	0.232	0.874	0.678	0.717
6	rs7685	0.361	0.956	0.977	0.628	0.529

Notes: ^aSelective variables were transformed into logarithmic model to normalize the distribution before analysis of covariance adjusted for age and gender. ^bNonparametric test for selective variables with non-normal distribution. ^cAnalysis of covariance adjusted for age and gender for selective variables with normal distribution.

Abbreviations: GARP, glycoprotein A repetitions predominant; AR, allergic rhinitis; SNP, single nucleotide polymorphism; Der p, Dermatophagoides pteronyssinus; Der f, Dermatophagoides farinae.

Table 8 Associations Between PER Risk and the Frequencies of Haplotypes

Haplotype	Haplotype Frequencies		Adjusted OR (95% CI)^a
rs1320646-rs3781699	Case	Control
G-A	0.581	0.529	1.000 (reference)
G-C	0.326	0.362	0.819 (0.676–0.993)
A-A	0.093	0.109	0.777 (0.573–1.050)

Notes: ^aDerived from logistic regression model performed by HAPSTAT 3.0 based on observed genotypes. Bold values denote statistical significance at the P < 0.05 level.

Abbreviation: PER, persistent allergic rhinitis; OR, odds ratio; CI, confidence interval.

Figure 1 Linkage disequilibrium (LD) of six SNPs in the GARP gene. LD of six SNPs was determined using the solid spine of LD option of Haploview 4.2. D’ values are displayed in the squares. Empty red squares have a pairwise D’ of 1.0. Red squares indicate strong pairwise LD, gradually coloring down to white squares of weak pairwise LD.

Table 9 Functional Annotation of 3 SNPs in silico Analysis

Chr: Position^a	Variant	Score^b	Enhancer Histone Marks	Motifs Changed	GRASP QTL Hits	Selected eQTL Hits	dbSNP Func Annot
11: 76659003	rs1320646	1f	7 tissues	7 altered motifs	2 hits	3 hits	3ʹUTR
11: 76658741	rs3781699	3a	8 tissues	Hltf	–	3 hits	3ʹUTR
11: 76660374	rs79525962	4	7 tissues	7 altered motifs	2 hits	3 hits	Missense

Notes: ^aBased on NCBI build 38 of the human genome. ^bBased on RegulomeDB (https://www.regulomedb.org/regulome-search/).

Abbreviation: SNP, single nucleotide polymorphisms; GRASP, Genome-Wide Repository of Associations Between SNPs and Phenotypes; eQTL, expression quantitative trait locus; UTR, untranslated region.

Figure 2 In silico prediction of secondary structures and minimum free energy (MFE) changes corresponding to GARP SNPs. (A) The MFE changed from −21 kcal/mol to −19.1 kcal/mol caused by rs79525962 (C > T), indicating a stronger binding affinity of microRNAs to the C allele. (B) The MFE changed from −16.4 kcal/mol to −21.6 kcal/mol caused by rs3781699 (A > C), indicating a stronger binding affinity of microRNAs to the C allele. (C) No change was found in MFE caused by rs1320646.

Abbreviation: GARP, glycoprotein A repetitions predominant.