1. The rare IL-13 R105Q variant, strongly enhances IL-13 protein activity David Rosenbaum 1, Gregory Hawkins 2, Lingxiang Zhu 3, Yin Chen 3, Eugene Bleecker 2, Deborah Meyers 2, Donata Vercelli 1 1. Arizona Respiratory Center, University of Arizona, Tucson, AZ, United States. 2. Center for Human Genomics, Wake Forest University, Winston-Salem, NC, United States. 3. Department of Pharmacology & Toxicology, University of Arizona, Tucson, AZ, United States. Abstract Because common genetic variants account for only a modest proportion of human complex disease risk, rare variants are currently under investigation as potential sources of phenotypic variance. Focusing on IL13, one of the most replicated asthma/allergy susceptibility genes, we sought to assess the contribution of rare coding variants to disease risk by coupling sequencing-based variant discovery with in vitro studies of protein activity. Sequencing of IL13 exons in DNA from 96 non-Hispanic White and 96 African American asthmatics identified a rare non-synonymous variant (rs140828306, minor allele frequency 1.6% in African Americans) in exon 4 that results in a non- conservative arginine (R) → glutamine (Q) substitution at amino acid residue position 105, immediately upstream of a conserved IL-13 receptor binding domain. For functional studies, recombinant WT IL-13 and IL-13 R105Q were expressed in eukaryotic cells and quantified by ELISA adjusting for differential antibody recognition of the variant. IL-13 R105Q was significantly more potent than WT IL-13 in inducing STAT6 phosphorylation and CD23 expression in human monocytes, and transcription of allergy-associated genes in primary human epithelial cells grown at an air-liquid interface. Neither protein acted on human T cells. These results show that IL-13 R105Q is a gain-of-function variant that is likely to significantly enhance IL-13-dependent events in vivo. Figure 2. IL-13 R105Q is more active than WT IL-13 in (A) inducing STAT6 phosphorylation and (B) upregulating CD23 in primary human monocytes. Human PBMCs were stimulated with increasing concentrations of WT IL-13 (circles) or IL-13 R105Q (squares) for (A) 1 or (B) 48 hours. Cells were stained with fluorochrome conjugated mAbs and assessed by immunofluorescence, gating on the monocyte population by forward and side scatter. Shown is one representative experiment out of five. Figure 4. IL-13 R105Q fails to activate STAT6 in primary human T cells. T cells lack expression of functional IL-4Rα/IL-13Rα1 heterodimers. Like WT IL-13, IL-13 R105Q fails to signal through the IL-4Rα/γc complex expressed on T cells. PBMCs were stimulated with IL-4 (0.5 ng/ml), WT IL-13, or IL-13 R105Q (0.5 ng/ml) for 1 hour. Cells were then surface stained with PE-conjugated anti-CD3, stained intracellularly with Alexa Fluor 488–conjugated anti-STAT6(pY641), and analyzed by flow cytometry gating on live cells. The figure shows the results of one representative experiment out of two. CD3 STAT6pY641 Figure 3. Increased transcription of allergy-related epithelial genes by IL-13 R105Q. Primary airway epithelial cells grown at an air-liquid interface were stimulated with WT IL-13 (circles) or IL-13 R105Q (squares) for 24 hours. mRNA was assessed by reverse transcription qPCR. Fold induction was calculated relative to the GAPDH housekeeping gene. Shown is one representative experiment out of three. Introduction The Th2 cytokine IL-13 is a critical mediator of allergic inflammation and is sufficient to induce many key features of experimental asthma. Consistent with the role of IL-13 in asthma/allergy, associations have been detected between IL13 common polymorphisms and disease phenotypes. These common variants have replicable but only modest effects on asthma/allergy risk. Low- frequency coding variants with medium-high penetrance are predicted to contribute to complex disease risk. To identify rare functional coding variants within IL13, exons from 96 non-Hispanic White and 96 African American severe asthmatics were sequenced. A rare variant particularly represented in African Americans was identified (rs140828306, minor allele frequency 1.6%). This non-synonymous rare variant is predicted to result in the non-conservative replacement of a polar basic arginine (R) with a polar uncharged glutamine (Q) at amino acid residue position 105 (R105Q). Moderate evolutionary conservation at amino acid residue 105 and the substitution’s proximity to a highly conserved IL-13 receptor binding domain suggest possible functional consequences, thereby potentially modifying susceptibility to asthma/allergy. Figure 1. A, Schematic of the IL13 gene structure with location of the single nucleotide polymorphism (SNP) found in African American asthmatics. SNP position is relative to IL13 ATG. B, Multi-species alignment for IL-13 shows moderate conservation at amino acid residue 105 (red box). Black arrows indicate amino acid residues involved in IL-13 receptor binding. C, Overview of experimental strategy. The correction factor was developed by expressing IL-13 (WT and variant) with [35 S] labeled methionine using the Thermo Scientific Pierce Human In Vitro Protein Expression System. Relative amounts of WT and variant protein were determined by SDS- PAGE/autoradiography/densitometry and used to adjust for differences detected by ELISA. D, Schematic representation of the IL-13 receptor complex and signaling pathway. Results IL-13 C rs140828306 +1923A → G MockIL-4 WT IL-13IL-13 R105Q D 105 Express recombinant WT IL-13 and IL-13 R105Q in eukaryotic cells. Develop correction factor for misrepresentation of IL-13 R105Q concentrations by an ELISA originally developed to detect WT IL-13. Determine recombinant protein concentrations. Quantitatively compare biological activity of WT IL-13 with IL-13 R105Q by stimulating primary human immune/epithelial cells. STAT6 IL-4R  IL-13R  1 IL-13 JAK-1Tyk 2 P P CD23 A B Conclusion A rare IL13 variant was identified in African American asthmatics. This polymorphism is predicted to result in a non-conservative R → Q substitution at amino acid residue position 105. This substitution occurs immediately next to an IL-13 receptor binding domain. In vitro functional studies show that IL-13 R105Q is significantly more active than WT IL-13 in enhancing effector pathways of allergic inflammation. IL-13 R105Q fails to engage primary human T cells. While the mechanisms underlying the increased potency of IL-13 R105Q require further investigation, and genotyping in well phenotyped populations is still in progress, our data strongly suggest IL-13 R105Q is a gain-of-function variant that may significantly enhance IL-13- dependent events in vivo, thereby modifying susceptibility to asthma/allergy. 0.4% 66.1% 3.3% 9.1% 53.4% 15.9% 5.6% 65.5% 4.0% 4.3% 66.1% 2.6% HYP6P.268 1945385