Selene M. Clay

Background: Genome-wide association studies of asthma have revealed robust associations with variation across the human leukocyte antigen (HLA) complex with independent associations in the HLA class I and class II regions for both childhood-onset asthma (COA) and adult-onset asthma (AOA). However, the specific variants and genes contributing to risk are unknown.Methods: We used Bayesian approaches to perform genetic fine-mapping for COA and AOA (n=9432 and 21,556, respectively;n=318,167 shared controls) in White British individuals from the UK Biobank and to perform expression quantitative trait locus (eQTL) fine-mapping in immune (lymphoblastoid cell lines,n=398; peripheral blood mononuclear cells,n=132) and airway (nasal epithelial cells,n=188) cells from ethnically diverse individuals. We also examined putatively causal protein coding variation from protein crystal structures and conducted replication studies in independent multi-ethnic cohorts from the UK Biobank (COAn=1686; AOAn=3666; controlsn=56,063).Results: Genetic fine-mapping revealed both shared and distinct causal variation between COA and AOA in the class I region but only distinct causal variation in the class II region. Both gene expression levels and amino acid variation contributed to risk. Our results from eQTL fine-mapping and amino acid visualization suggested that theHLA-DQA1*03:01 allele and variation associated with expression of the nonclassicalHLA-DQA2andHLA-DQB2genes accounted entirely for the most significant association with AOA in GWAS. Our studies also suggested a potentially prominent role for HLA-C protein coding variation in the class I region in COA. We replicated putatively causal variant associations in a multi-ethnic cohort.Conclusions: We highlight roles for both gene expression and protein coding variation in asthma risk and identified putatively causal variation and genes in the HLA region. A convergence of genomic, transcriptional, and protein coding evidence implicates theHLA-DQA2andHLA-DQB2genes andHLA-DQA1*03:01 allele in AOA.

Previous genotype:phenotype mapping of the mouse and primate dentition revealed the presence of pre- and post-canine modules in mice and anthropoid primates, as well as molar and premolar submodules in anthropoid primates. We estimated phenotypic correlation matrices for species that sample broadly across Mammalia to test the hypothesis that these modules exist across a broader range of taxa and thereby represent a conserved mammalian trait. We calculated phenotypic correlation matrices from linear dental measurements of 419 individual specimens representing 5 species from 4 mammalian orders: Artiodactyla (Odocoileus hemionus), Carnivora (Canis latrans and Ursus americanus), Didelphimorphia (Didelphis virginiana), and Primates (Colobus guereza). Our results based on hierarchical clustering indicate a generally higher correlation within incisors and among post-canine teeth. However, the post-canine phenotypic correlation matrices do not consistently exhibit the premolar and molar submodularity observed in anthropoid primates. Additionally, we find evidence of sex differences in the Odocoileus phenotypic correlation matrices: Males of this species exhibit overall higher inter-trait correlations compared to females. Our overall findings support the interpretation that incisors and post-canine dentition represent different phenotypic modules, and that this architecture may be a conserved trait for mammals.

Mononuclear phagocytes (MP) have central importance in innate immunity, inflammation, and fibrosis. Recruited MPs, such as macrophages, are plastic cells and can switch from an inflammatory to a restorative phenotype during the healing process. However, the role of the MPs in corneal wound healing is not completely understood. The purpose of this study is to characterize the kinetics of recruited MPs and evaluate the role of macrophage metalloelastase (MMP12) in the healing process, using an in vivo corneal chemical injury model. Unwounded and wounded corneas of wild-type (WT) and Mmp12^−/− mice were collected at 1, 3, and 6 days after chemical injury and processed for flow cytometry analysis. Corneal MP phenotype significantly changed over time with recruited Ly6C^high (proinflammatory) cells being most abundant at 1 day post-injury. Ly6C^int cells were highly expressed at 3 days post-injury and Ly6C^neg (patrolling) cells became the predominant cell type at 6 days post-injury. CD11c⁺ dendritic cells were abundant in corneas from Mmp12^−/− mice at 6 days post-injury. These findings show the temporal phenotypic plasticity of recruited MPs and provide valuable insight into the role of the MPs in the corneal repair response, which may help guide the future development of MP-targeted therapies.

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as an enhancer-blocking element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that the asthma-associated single nucleotide polymorphism (SNP) rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a mechanism through which a regulatory SNP contributes to genetic risk of asthma.

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as a strong regulatory element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that genotype at the asthma-associated SNP rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a novel mechanism through which a regulatory SNP contributes to genetic risk of asthma.

Following corneal injury, coordinated cellular and protein interactions occur at the wound site to restore tissue homeostasis. Regulation of this response is required to prevent the development of chronic inflammation, abnormal neovascularization, and fibrosis. The chemokine CCL2 and its primary receptor CCR2 are key regulators of the inflammatory and neovascular responses to injury. In this study, we investigated the role of macrophage-associated matrix metalloproteinase 12 (MMP12) in the regulation of CCL2 and CCR2 after corneal wounding. Using two corneal injury models, we examined the temporal and spatial expression of CCL2 and CCR2 in Mmp12^−/− and wild-type (WT) mice. Our data showed that MMP12 downregulated CCL2 and CCR2 expression in a manner dependent on the timing and mechanism of injury. We also examined the effect of CCL2 on the injury response in Mmp12^−/− and WT corneas. We found that macrophage infiltration and neovascularization following CCL2 blockade was significantly reduced in Mmp12^−/− corneas as compared with WT corneas. These findings indicate that MMP12 inhibits corneal inflammation and neovascularization after injury through its regulation of CCL2.