A Genome Wide Association Study of Plasma Total IgE Concentration in the Framingham Heart Study

Introduction

Immunoglobulin E (IgE)-mediated allergy to environmental allergens plays a central role in the pathophysiology of asthma, allergic rhinoconjunctivitis, atopic dermatitis, and food allergy. Atopic persons have abnormally high concentrations of IgE reactive against one or more specific allergens and often have an elevated plasma total IgE concentration.¹ Atopy and elevated IgE concentrations are genetically complex traits, and the specific genetic risk factors that lead to IgE dysregulation and clinical atopy are an area of active investigation.

Genetic linkage studies of total IgE levels have been conducted in numerous samples ascertained for asthma and atopy with associations reported for locations on multiple chromosomes. Fine mapping of genes at several of these locations as well as candidate gene studies have implicated a number of genetic variants as potentially important determinants of plasma IgE concentration, with IL-4, IL-13, and STAT6 among the most consistently replicated associations.^2-3

To date, there have been two genome-wide association studies (GWAS) published investigating total IgE concentration. Weidinger et al.⁴, in 2008, reported findings from a population-based, German cohort of 1,530 individuals, with replication analyses performed in four independent population-based study samples that included a total n=9,769 subjects. The study found that functional variants of the alpha chain of the high affinity receptor for IgE (FCER1A) were strongly associated with total IgE levels. The study also confirmed STAT6 as a susceptibility locus as well as identifying RAD50, located adjacent to the IL-13 gene, as a potential determinant of IgE dysregulation. More recently, the GABRIEL asthma genetics consortium found, among both asthmatic cases and non-asthmatic controls, a SNP near HLA-DRB1 to be associated with total IgE with genome-wide statistical significance, as well as evidence of association for FCER1A, STAT6, and IL-13.⁵

In this study, we report the results of a GWAS of plasma total IgE concentration in the Framingham Heart Study. In addition to confirming FCER1A, STAT6, and IL-13 as susceptibility genes for IgE dysregulation, we identify genetic variants in HLA genes as potential determinants of atopy and IgE concentration.

Methods

Results

Characteristics of the FHS participants included in the IgE GWAS are provided in Table 1, and characteristics of replication cohorts along with FHS participants are provided in Table E1 in the Online Repository. A Manhattan plot of the GWAS findings is shown in Figure 1. The Q-Q plot (Figure E1) does not show strong departure from the distribution of expected p-values by chance, with a genomic control parameter of 1.029, suggesting that familial relationships and population stratification were appropriately controlled for in the analysis.

In the FHS GWAS, 13 SNPs met genome-wide significance as determined by the commonly accepted significance threshold of 5 × 10^-08.¹⁵ All 13 SNPs were within 60 kb of three genes, FCER1A, STAT6, and IL-13. Details of these 13 SNPs, as well as all 527 SNPs with a nominal p-value < 10 ^-4, can be seen in Figures E2-E4 and Table E2, respectively, in the Online Repository.

Of the top 70 SNPs selected for the meta-analysis with replication cohorts (Table E3), 10 SNPs demonstrated replication based on a Bonferroni-corrected alpha criterion of p < 0.05 / 70 = 0.0007 in a meta-analysis of the replication cohorts. (Table 2). Two additional SNPs, near the gene regions, HLA-A and HLA-G, did not meet the p < 0.0007 criterion in the replication cohorts but had association p values meeting the genome-wide significance criterion of p < 5 × 10^-08 in meta-analysis of the replication cohorts together with the FHS discovery cohort (Table 2). Cohort-specific association results for these 12 SNPs are shown in Table E4. The top 2 SNPs, rs2251746 and rs2494264, located in non-coding regions of FCER1A, had combined (FHS plus replication cohorts) meta-analysis p-values of 4.52 × 10^-26 and 9.40 × 10^-20, respectively. SNPs rs1059513 in STAT6 and rs20541 in IL-13 were also associated with total IgE level at genome-wide significance, each with a combined meta-analysis p-value < 10^-11.

Among the genome wide-significant associations based on combined meta-analysis p-values were SNPs in or near the HLA-A, HLA-G, HLA-DQA2, and DARC genes (Table 2). Two SNPs within a single LD block near the HLA-A gene were found to have meta-analysis p-values < 5 × 10^-8 (http://www.broad.mit.edu.mpg/haploview). rs2571391 is located within 60 kb of the HLA-A gene and was the most highly associated SNP in this region, with a combined p-value of 1.23 × 10^-15. Association plots for the regions encompassing HLA-A and HLA-G (Figure E5) and HLA-DQA2 (Figure E6) depict the associations in these regions in FHS.

The SNP rs13962 is a missense SNP in the DARC gene and was associated with total IgE with a combined p-value of 2.21 × 10^-11. Given its close proximity to the FCER1A gene, a conditional model regression analysis was performed to assess the independence of this association. After adjusting for rs2251746, the top FCER1A SNP, the association of DARC was not found to be significant in FHS (p-value = 0.6). Similar findings were seen in all of the replication cohorts.

The GABRIEL consortium recently reported evidence of associations for both asthma and total IgE with SNPs in the HLA-DQB1 – HLA-DRB1 region.⁵ The total IgE peak was distinct from the asthma peak and best represented by rs9271300, which was not included in our meta-analysis because it is not part of the HapMap 2 panel. We were, however, able to impute rs9271300 with moderate accuracy (observed to expected variance ratio = 0.5) using the 1000 Genomes Project June 2010 haplotypes as a template.¹⁶ The association of this imputed SNP with log₁₀-transformed total IgE in the FHS dataset was not significant (p=0.06); however, the 95% confidence interval (-0.001 to +0.061) around the regression coefficient of 0.03 in FHS included the GABRIEL regression coefficient (0.06) for this association, suggesting that the findings are not heterogeneous with those of the GABRIEL study.

Discussion

We found that SNPs in three gene regions, FCER1A, STAT6, and IL-13, were associated with total IgE concentration at a genome-wide significant level in the FHS cohort. The results confirm the findings from prior studies that have implicated these genes in the pathogenesis of IgE dysregulation. Additionally, we observed associations with SNPs near three other genes, HLA-A, HLA-G, and HLA-DQA2, for which relationships to IgE have not been clearly established.

Our analysis reveals associations, which replicate in independent cohorts and/or reach genome-wide significance in meta-analysis combining FHS with replication cohorts, in the region of the MHC superlocus on chromosome 6. While MHC II antigens, particularly HLA-DRB1 and HLA-DQB1, have been consistently associated with atopic phenotypes in the genetic literature,^17-18 evidence for a genetic susceptibility via the classical MHC I antigens (HLA-A, -B, -C) is less clear. The stronger association of MHC II antigens versus MHC I antigens in the literature is consistent with their functional roles in the immune system, with MHC I responsible for initiating cell mediated immunity, targeting infected and cancer cells, and MHC II mediating humoral immunity, including the production of IgE antibodies to allergenic antigens.

Three SNPs in or near the HLA-A gene, an MHC I antigen, were found to have p-values < 2 × 10^-7 in FHS, with the highest association seen for rs2571391 with a combined (FHS plus replication cohorts) meta-analysis p-value of 1.23 × 10^-15. The first study to identify a linkage between HLA-A haplotypes and ragweed allergy was published in 1972 by Levine et al.,¹⁹ though a subsequent, larger study was unable to replicate the original findings.²⁰ In 1979, Marsh et al published a regression analysis evaluating total IgE and found both positive (HLA-A2) and negative (HLA-A3) associations among the HLA-A antigens in patients with varying degrees of ragweed sensitivity.²¹ More recent studies have been unable to find significant associations between HLA-A and total IgE,²² intrinsic or extrinsic asthma,²⁰ and mite sensitive allergy in Greek and Venezuelan populations.^23-24 However, results published online from the British 1958 birth cohort show associations (at conventional levels of significance) between log-transformed total IgE and directly measured HLA-A subtypes HLA*02, *03, and *29 [see URLs below]. (http://www.b58cgene.sgul.ac.uk/phenotype0.php?snp=hlaa_02&pheno=1) (http://www.b58cgene.sgul.ac.uk/phenotype0.php?snp=hlaa_03&pheno=1) (http://www.b58cgene.sgul.ac.uk/phenotype0.php?snp=hlaa_29&pheno=1)

In the clinical literature, many studies in children have demonstrated a relationship between viral infection, particularly RSV, and the later development of asthma and increases in specific IgE.^25-26 One hypothesis is that MHC I polymorphisms may influence IgE concentration through the processing of infectious pathogens, leading to immune dysregulation and, eventually, the clinical findings of atopy and asthma.

Our findings suggest that HLA-A may represent a susceptibility locus for IgE dysregulation. While both MHC I and MHC II loci were found to be associated with total IgE, the association of HLA-A was consistently stronger in both the original FHS GWAS and in the meta-analysis than the MHC II antigens, HLA-DQB1 and HLA–DRB1.

Additionally, a SNP near the HLA-G gene, a non-classical MHC I antigen that has been associated with asthma and bronchial hyper-responsiveness in a positional candidate gene study²⁷ was also found to reach genome wide significance in the combined meta-analysis. HLA-G has not previously been associated with total IgE dysregulation but is thought to have immunomodulatory effects via actions on NK cells, T lymphocytes, and antigen presenting cells.²⁸

The HLA-DQA2 region had the strongest association among MHC II loci, with rs2858331, a SNP within 27 kb from the HLA-DQA2 gene, observed at a genome-wide significant level in the combined meta-analysis (p-value = 1.44 × 10^-08). HLA-DQA2 is thought to play a central role in peptide loading of MHC II molecules. Though it has never been associated with total IgE in the literature, a genome-wide association study by Li et al. found a strong association of asthma with a nearby SNP, rs3916765, in the same region.²⁹

In the GABRIEL Consortium study,⁵ the rs9271300 SNP near HLA-DRB1 was significantly associated with total IgE and was the strongest association observed for this phenotype. As discussed previously, this SNP was not found to be strongly associated with total IgE in FHS. It is not clear why the association with HLA-DRB1 was considerably weaker in the FHS population compared to the GABRIEL population. This finding may be partly explained by the difference in study populations as well as the difficulty in discriminating separate signals from the MHC superlocus. This discordance could also reflect the imperfect imputation of rs9271300 in our study.

The DARC gene on chromosome 1, in close proximity to FCER1A, was also identified as a susceptibility locus in the meta-analysis. The DARC gene was of particular interest given the biologic plausibility of DARC as a modulator of the body’s inflammatory response and literature suggesting a role for DARC in the development of asthma and IgE dysregulation. A recent study by Vergara et al investigated the relationship between DARC and asthma phenotypes in subjects of African descent. A SNP in the DARC promoter region, rs2814778, located in the same LD block as the top DARC SNP in FHS (http://www.broad.mit.edu.mpg/haploview), was found to be significantly associated with total IgE in Colombian and African Caribbean populations and with asthma in Colombian, Brazilian, and African Caribbean populations.³⁰

Based on the HapMap CEPH sample, there is low to moderate LD between the highest associated SNP in DARC, rs13962, and the nearby top SNP in FCER1A (rs2251746) with r² = 0.27. (Figure E1) (http://broad.mit.edu/mpg/snap) Conditional regression analysis, performed in FHS and the 5 independent replication cohorts, revealed that the association of DARC with total IgE concentration was not significant once rs2251746 was included in the model. While this suggests that the DARC association in the Vergara study may be confounded by the effect of the neighboring FCER1A gene, differences in the populations studied in this meta-analysis (predominantly Caucasian of European descent) and the Vergara study (African descent) likely play a role as the minor allele frequencies of the DARC SNP are significantly higher in populations of African descent compared to populations of European descent. Given the known function of the DARC receptor to act as a chemokine sink and modulate the inflammatory cascade,³¹ the DARC associations with asthma and total IgE remain plausible, particularly in African populations. Further studies to better delineate the roles of DARC and FCER1A in IgE dysregulation are warranted.

Our study also confirmed, at a genome-wide significant level, the findings from prior genetic literature implicating FCER1A, STAT6, and IL13 as susceptibility loci for IgE dysregulation. In the first published GWAS of total IgE concentration, Weidinger and colleagues identified FCER1A as a susceptibility gene in a population based German cohort.⁴ The top FCER1A SNP in the Weidinger study, rs2427837, was also highly associated with total IgE in FHS and had the second lowest p-value among all top SNPs (p=9.82 × 10^-12). STAT6 and IL-13 are known to play significant roles in the pathogenesis of IgE dysregulation. IL-13, in conjunction with IL-4, is a critical mediator of the T_H2 allergic response and STAT6 is considered the master regulator of IgE class switch recombination.^32-33 Our observation of a strong association of STAT6 SNPs with IgE concentration confirms previous linkage and candidate gene studies which have shown STAT6 genetic variants to be associated with food allergy,³⁴ atopic dermatitis,³⁵ asthma,³⁶ and total IgE.³⁷ In a recent review of asthma and atopy genetics, Ober and Hoffman identified IL-13 as one of 8 “elite” genes which had been associated with an asthma or atopy phenotype in greater than 10 studies.³

The RAD50 gene is located within 20 kb of IL-13 on chromosome 5. In the Weidinger study,⁴ as well as the subsequent GWAS investigating asthma by Li and colleagues,²⁹ the RAD50 gene was found to be more highly associated with total IgE and asthma, respectively, than the neighboring IL-13 gene. This is in contrast to the FHS cohort in which SNPs located in IL-13 were found to have the strongest associations in the region, with no SNPs within the RAD50 gene found to have a p-value < 10^-4 (see Figure E3). The highest associated SNPs in each (rs1295686 for IL-13, rs2240032 for RAD50) were in moderate LD with an r²=0.58 in the HapMap CEPH sample. Given the findings from three separate GWAS, it is difficult to separate the two signals on a genetic level. With mouse models showing a T_H2 locus control region in the 3’ end of RAD50, ³⁸ both Il-13 and RAD50 are plausible candidate genes in the pathogenesis of IgE dysregulation and asthma.

In summary, this GWAS of plasma total IgE in the Framingham Heart Study has identified potential susceptibility loci in the HLA-A, HLA-G, HLA-DQA2 gene regions for which an association with total IgE has not been clearly established in the previous literature. We have also confirmed the associations of previously described genetic variants in FCER1A, STAT6, and IL-13 as risk factors for IgE dysregulation. Based on these findings, further genetic investigation of the relationship between HLA loci and total IgE and other atopy phenotypes is warranted. These findings may help target molecular pathways for future interventions to prevent or treat IgE-mediated allergy.