STAT6 and LRP1 polymorphisms are associated with food allergen sensitization in Mexican children

To the Editor:

Food allergy, affecting up to 8% of children in the United States,¹ is most often mediated by allergen specific immunoglobulin E (IgE) antibodies in the blood. Food allergen sensitization is an intermediate phenotype measured by specific IgE blood tests or skin prick tests (SPTs) and used in the diagnosis of food allergy. Despite family history being a risk factor, no genetic variants have been conclusively identified for food sensitization or clinical food allergy.² Using the Mexico City Childhood Asthma Study (MCCAS), we examined associations between food sensitization (based on SPTs) and single nucleotide polymorphisms (SNPs) spanning five autosomal candidate genes reviewed by Hong et al. [CD14 (cluster of differentiation 14), IL10 (interleukin 10), IL13 (interleukin 13), SPINK5 (serine peptidase inhibitor, Kazal type 5 isoform), and STAT6 (signal transducer and activator of transcription)].²

We previously conducted a genome-wide association study of asthma in MCCAS among 492 children with physician-diagnosed asthma (aged 5-17) and their parents, who were recruited from a pediatric allergy clinic in Mexico City.³ The children’s clinical evaluation included SPTs to six major food allergens that are common in the Mexican diet (milk, egg, wheat, soy, peanuts, and tree nuts). A SPT was declared positive if the largest diameter of the wheal exceeded 4mm. Testing was considered valid if the reaction to the positive control (histamine) exceeded 6 mm and also exceeded 4 mm above the negative control (glycerin).⁴ There were 162 trios having an asthmatic child with a positive SPT to at least one food allergen. We examined SNP associations with food sensitization in the five candidate genes in these 162 trios.² As all probands are asthmatics, it is not possible to adjust for asthma. These associations are generalizable to the asthmatic population, but not necessarily to the general population.

Genotyping was performed on the Illumina HumanHap 550v3 BeadChip, and standard quality control filters were applied.³ For this analysis, we selected SNPs spanning five candidate gene regions (20kb upstream of the 5′ end through 20kb downstream of the 3′ end), resulting in 343 SNPs (16 CD14, 52 IL10, 39 IL13, 28 STAT6, and 208 SPINK5 SNPs). SNPs were directly genotyped or imputed using MaCH (http://www.sph.umich.edu/csg/abecasis/MACH/index.html) with HapMap phase II release 21 reference haplotypes combined from CEU (European Americans), YRI (African Americans), and CHB+JPT (Chinese+Japanese).

PBAT v3.61 (http://www.biostat.harvard.edu/~clange/default.htm) was used to conduct family-based association tests on additive SNP genotype dosage values (estimated reference allele counts with a fractional value ranging from 0 to 2.0). To correct for multiple testing, Bonferroni correction for the total number of SNPs (P<α/M, where M is the number of independent tests) is too conservative, given our observed correlation patterns among SNPs. Therefore, we applied the widely-used Nyholt correction (P<α/M_eff), which adjusts for the effective number of independent tests (M_eff) based on the correlation matrix of pairwise LD among the SNPs.^5,6 This method gave a significance threshold of P<0.0011 A less conservative significance threshold based on the number of candidate genes examined is P<0.01. Association results and LD patterns were plotted together using SNAP (http://www.broadinstitute.org/mpg/snap/).

In the 162 trios with a food allergen sensitized child, no SNP associations were statistically significant at P<0.0011. However, three genotyped SNPs in or near STAT6 were associated with food sensitization at P<0.01 (Table I), two intronic SNPs in the nearby LRP1 (low density lipoprotein receptor-related protein 1) gene and a 3′ untranslated STAT6 SNP. Their minor allele frequencies were lower in the food sensitized children (39.5% for the top SNP rs4759044) compared to the children with no sensitization to the six food allergens (42.2% for rs4759044). The STAT6 and LRP1 SNPs are in moderate LD in CEU (Figure 1, r²=0.34-0.37) and weaker LD in YRI (r²=0.08-0.24) and CHB+JPT (r²=0.12-0.13). Of 28 SNPs spanning the STAT6 region, none were associated with asthma at P<0.05, despite a three-fold greater sample size and thus more statistical power (Table I). There was minimal evidence for SNP associations with food sensitization across CD14, IL10, IL13 or SPINK5 (results not shown).

Table I.

Associations of the 28 SNPs examined in or near STAT6 (±20kb) in 492 trios with an asthmatic child and the subset of 162 trios with a food sensitized child (at least one positive skin prick test to six major food allergens). The flanking regions around STAT6 included SNPs in two other genes, NAB2 and LRP1. SNPs were genotyped or imputed, and MaCH Rsq values. indicate imputation quality.

SNPa	SNP Type	Gene	MAFb	MaCH Rsq value	P, asthma (N=492 trios)	P, food sensitization (N=162 trios)
rs17119418	intergenic	-	0.050	0.77	0.085	0.19
rs2035545	intergenic	-	0.053	0.80	0.19	0.17
rs324010	intergenic	-	0.41	0.92	0.035	0.010
rs324020	intronic	NAB2	0.48	0.94	0.034	0.020
rs1059513c	intergenic	-	0.074	0.99	0.35	0.56
rs703817c	3′ UTR	STAT6	0.47	0.99	0.026	0.0076
rs324015c	3′ UTR	STAT6	0.31	0.99	0.28	0.084
rs3024975	intronic	STAT6	0.060	0.81	0.25	0.30
rs3024974	intronic	STAT6	0.060	0.80	0.24	0.29
rs841718c	intronic	STAT6	0.44	0.99	0.072	0.015
rs3024971	intronic	STAT6	0.029	0.88	0.077	0.10
rs10783813	intronic	STAT6	0.20	0.61	0.57	0.23
rs324011	intronic	STAT6	0.47	0.97	0.044	0.055
rs167769c	intronic	STAT6	0.47	0.99	0.044	0.029
rs324013c	intergenic	-	0.39	0.99	0.31	0.41
rs12368672	intergenic	-	0.44	0.90	0.082	0.066
rs11172106	intergenic	-	0.41	0.91	0.28	0.36
rs12298170	intergenic	-	0.41	0.88	0.27	0.31
rs12307379	intergenic	-	0.020	0.69	0.21	0.011
rs7306742	intergenic	-	0.020	0.70	0.21	0.012
rs11172113c	intronic	LRP1	0.40	0.99	0.24	0.21
rs4759044c	intronic	LRP1	0.34	0.99	0.25	0.0056
rs1385526	intronic	LRP1	0.49	0.88	0.24	0.019
rs715948c	intronic	LRP1	0.27	0.99	0.32	0.011
rs4759277	intronic	LRP1	0.41	0.97	0.44	0.066
rs1466535c	intronic	LRP1	0.43	0.99	0.17	0.0077
rs7968719	intronic	LRP1	0.41	0.76	0.18	0.11
rs7398375	intronic	LRP1	0.30	0.66	0.37	0.034

^aSNPs ordered by their position on chromosome 12.

^bMAF among the 162 food allergen sensitized children.

^cGenotyped SNPs.

SNP, single nucleotide polymorphism; MAF, minor allele frequency; UTR, untranslated region

Figure 1.

Association results and linkage disequilibrium patterns of SNPs in or near STAT6 (±20kb). Correlations between the SNP with the lowest P value (rs4759044) and surrounding SNPs are shown with reference to the HapMap CEU population, with darker red shading indicating higher r² values. Gene annotations are shown in green, and estimated recombination rates are shown in light blue.

Another 3′ untranslated STAT6 SNP (rs324015) was previously associated with risk for, and severity of, nut allergy in white atopic children from the United Kingdom,⁷ but this association was not corroborated for severity of food allergy in Japanese children.⁸ In our study of Mexican children, rs324015 gave a P value of 0.072, but its adjacent SNP (rs703817) was associated with food sensitization at P=0.0076. The implicated SNPs may tag underlying functional or regulatory variants, and it is not surprising that association patterns vary across populations with dissimilar LD patterns.

LRP1 and its protein product have been largely implicated in neurologic processes. STAT6 SNPs have been associated with food allergy,^7,8 asthma,⁹ total IgE,¹⁰ and eosinophilic esophagitis.¹¹ STAT6 plays a central role in mediating IL4 (interleukin 4) and IL13 signals for IgE antibody production.² Given its biological role, STAT6 polymorphisms are more likely than LRP1 polymorphisms to influence food sensitization. Nonetheless, the association patterns between STAT6 and LRP1 SNPs reflect moderate LD with low recombination across the region (Figure 1). While our findings might not be generalizable to the general population, we provide evidence that STAT6 and LRP1 polymorphisms are associated with food sensitization in asthmatics, who are at particularly high risk for developing sensitization. Follow-up studies in Hispanics and other ethnicities could help to refine these associations with food sensitization and clinical food allergy.

Abbreviations

CD14

cluster of differentiation 14

CEU

Utah residents with Northern and Western European ancestry from the Centre d’Etude du Polymorphisme Humain collection (HapMap population)

CHB

Han Chinese (HapMap population)

IgE

immunoglobulin E

IL10

interleukin 10

IL13

interleukin 13

JPT

Japanese (HapMap population)

LD

linkage disequilbrium

LRP1

low density lipoprotein receptor-related protein 1

MAF

minor allele frequency

MCCAS

Mexico City Childhood Asthma Study

SNP

single nucleotide polymorphism

SPINK5

serine peptidase inhibitor, Kazal type 5 isoform

SPT

skin prick test

STAT6

signal transducer and activator of transcription 6

UTR

untranslated region

YRI

Yoruban (HapMap population)