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Whole Genome Sequencing Identifies Four Novel Variants in the Epidermal Differentiation Complex That Increase Risk and Severity for Atopic Dermatitis
269
Hox5 paralogous genes modulate Th2 cell function during chronic allergic inflammation via regulation of Gata3
Catherine Ptaschinski, Steven M. Hrycaj, Leilani Marty Santos, Matthew A. Schaller, Deneen M. Wellik, and Nicholas W. Lukacs; University of Michigan, Ann Arbor, MI. RATIONALE: The HOX5 proteins (HOXA5, HOXB5 and HOXC5) are important in lung development, however our preliminary results indicate that these genes continue to function beyond the embryonic stage. We therefore asked whether they contribute to the development of allergic airway disease in adult mice. METHODS: Hox5 mutant mice and WT littermate controls were treated with cockroach allergen (CRA) and lung inflammation was measured by flow cytometry and histology. Mediastinal lymph node (MLN) cells were restimulated with CRA and cytokine production was measured by bioplex. T cell studies were performed using na€ıve T cells from spleens or the human Jurkat T cell line. RESULTS: Hox5 mutant mice have increased lung inflammation following CRA exposure, including increased numbers of CD4+ T cells. Cells from the MLN of these mice produce significantly more Th2 cytokines when restimulated. These mice also produced more mucus in the lungs compared to WT mice. These results were replicated using bone marrow chimeras, in which mice that received Hox5 mutant bone marrow demonstrated exacerbated Th2 pathology compared to mice that received WT bone marrow, indicating a primary role for the hematopoietic system. When na€ıve T cells were skewed to Th2 cells, Hox5 mutant cells produced more Th2 cytokines and expressed more Gata3 than WT cells. Expression of HOX5 protein in Jurkat cells resulted in binding to the Gata3 gene at known STAT6 sites, indicating cooperation between these transcription factors in Gata3 gene regulation. CONCLUSIONS: These results indicate a novel role for HOX5 proteins as regulators of Th2 cell differentiation during allergic disease.
270
Whole Genome Sequencing Identifies Four Novel Variants in the Epidermal Differentiation Complex That Increase Risk and Severity for Atopic Dermatitis
Rasika A. Mathias1, Sameer Chavan2, Meher Boorgula3, William O. C. Cookson, MD, DPhil4, Saffron Willis-Owen5, Nicholas M. Rafaels6, Jon M. Hanifin, MD, FAAAAI7, Amy Paller8, Lynda C. Schneider, MD, FAAAAI9, Richard Gallo, MD, PhD10, Emma Guttman-Yassky, MD, PhD11, Peck Y. Ong, MD, FAAAAI12, Ingo Ruczinski13, Terri Beaty1, Li Gao1, Lisa A. Beck, MD, FAAAAI14, Miriam Moffat15, Donald Y. M. Leung, MD, PhD, FAAAAI16, and Kathleen C. Barnes, PhD, FAAAAI17; 1Johns Hopkins, BALTIMORE, 2University of Colorado, denver, 3University of Colorado, DENVER, 4Imperial College London, London, United Kingdom, 5Imperial College, LONDON, United Kingdom, 6Department of Medicine, University of Colorado, Denver, 7Oregon Health and Science University, Portland, OR, 8Northwestern University, CHICAGO, 9Harvard Medical School, Boston, MA, 10Division of Dermatology, University of California, San Diego, San Diego, CA, 11 Mount Sinai Health System - Dermatology, New York, NY, 12Children’s Hospital Los Angeles/USC, Los Angeles, CA, 13Johns Hopkins University, BALTIMORE, 14Department of Dermatology, University of Rochester Medical Center, Rochester, NY, 15Imperial College, LONDON, 16 K926i, National Jewish Health, Denver, CO, 17University of Colorado Denver, Aurora, CO. RATIONALE: Filaggrin (FLG) mutations located within the Epidermal Differentiation Complex (EDC; chr1:151972910-153642037) are known to increase risk for atopic dermatitis (AD). However, the role of variants within additional EDC genes is poorly understood. METHODS: We use whole genome sequencing on 493 European American AD subjects (average age 5 26, average severity measured by
EASI score 5 14.5) and 237 non-atopic controls in the Atopic Dermatitis Research Network (ADRN) study to test for association with single nucleotide polymorphisms (SNPs) in the EDC as risk determinants of AD as well as severity of disease. Replication was performed in an independent European sample of 609 AD cases and 695 controls. RESULTS: We identified 12 SNPs with a p <0.001 comparing AD to nonatopic controls including the well documented stop gain mutation in FLG (rs61816761, p 5 0.0003). Four of these SNPs were replicated in an independent European population (p<0.00001) including the stop gain mutation in FLG, two intergenic SNPs and one noncoding RNA (FLGAS1). Odds ratios for all SNPs were >3 in the ADRN dataset and ranged from 2.5-3 in the replication dataset. Severity of disease as measured by total serum IgE, eosinophil count, phadiatop and EASI score in all the AD subjects from the ADRN study increased with carrier status (i.e. AD subjects with all four risk alleles were more severe compared to those with 0-3 risk alleles, p<0.05). CONCLUSIONS: We have identified for the first time that non FLG variants in the EDC could result in added burden for risk and increased severity of AD.
271
A Cross-Sectional Study of Asthma Exacerbations in the Emergency Department: Viral Infections and the Microbiome
Joshua Kennedy, MD1,2, Olga Hardin, MD3, Jesse Denson, PhD4, Kurt Schwalm, BS5, Suzanne Godbold, RRT, AE-C3, Lee Crawley, MS, RRT-NPS3, Thomas Abramo, MD6, Ashley N. Stoner, MD3, John C. Kincaid, MD7, and Darrell L. Dinwiddie, PhD5; 1Arkansas Children’s Research Institute, Little Rock, AR, 2Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR, 3University of Arkansas for Medical Sciences, Little Rock, AR, 4University of New Mexico, Albuquerque, NM, 5Department of Pediatrics, University of New Mexico HSC, Albuquerque, NM, 6university of Arkansas for Medical Sciences, Little Rock, AR, 7University of Arkansas for Medical Sciencess, Little Rock, AR. RATIONALE: We employ next-generation sequencing to evaluate viral genomes and microbiomes of children with asthma exacerbations and controls in order to compare clinical markers of disease severity. METHODS: We enrolled children from the emergency department with asthma exacerbations and children with cold symptoms (controls). We obtained nasal pharyngeal swabs, Asthma Control Tests (ACT), and modified Jackson criteria (MJC). To identify viral infections, we used targeted RNA hybridization-based genome detection methods that included 34 respiratory viruses. We sequenced the variable regions 4-6 of 16S ribosomal RNA gene. Clinical data was compared between viralinduced asthma, asthma, viral-induced colds, and colds. Microbiome data was used to determine if specific bacteria or diversity were associated with viral-induced wheezing. RESULTS: ACT scores were lower in asthmatics with virus compared to asthmatics without virus (med517 vs. 20, respectively; p<0.05). Viralinduced asthmatics had worse lower respiratory symptoms by MJC than viral-induced colds control (med58 and 4, respectively; p<0.05). We identified five microbiome profiles classified by dominant genus, Moraxella, Corynebacterium, Staphylococcus, Haemophilus, and Diverse. Controls were Moraxella-dominant in 52.9% and Diverse in 17.6%. Asthmatics were 27.6% Moraxella-dominant and 37.9% Diverse. The microbiome of asthmatics with respiratory syncytial virus (RSV) infection trended towards a greater number of species than the microbiome from subjects with other viruses (mean 362 (RSV) vs. 276 (Other viruses); p50.055). CONCLUSIONS: Viral-induced asthma exacerbations are more likely to occur in children with less overall asthma control. Lower respiratory symptoms are more pronounced in asthmatics with viruses than in controls. Asthmatics tend to have a more diverse nasopharyngeal microbiota.
SATURDAY
Abstracts AB85
J ALLERGY CLIN IMMUNOL VOLUME 139, NUMBER 2
Hox5 paralogous genes modulate Th2 cell function during chronic allergic inflammation via regulation of Gata3
Catherine Ptaschinski, Steven M. Hrycaj, Leilani Marty Santos, Matthew A. Schaller, Deneen M. Wellik, and Nicholas W. Lukacs; University of Michigan, Ann Arbor, MI. RATIONALE: The HOX5 proteins (HOXA5, HOXB5 and HOXC5) are important in lung development, however our preliminary results indicate that these genes continue to function beyond the embryonic stage. We therefore asked whether they contribute to the development of allergic airway disease in adult mice. METHODS: Hox5 mutant mice and WT littermate controls were treated with cockroach allergen (CRA) and lung inflammation was measured by flow cytometry and histology. Mediastinal lymph node (MLN) cells were restimulated with CRA and cytokine production was measured by bioplex. T cell studies were performed using na€ıve T cells from spleens or the human Jurkat T cell line. RESULTS: Hox5 mutant mice have increased lung inflammation following CRA exposure, including increased numbers of CD4+ T cells. Cells from the MLN of these mice produce significantly more Th2 cytokines when restimulated. These mice also produced more mucus in the lungs compared to WT mice. These results were replicated using bone marrow chimeras, in which mice that received Hox5 mutant bone marrow demonstrated exacerbated Th2 pathology compared to mice that received WT bone marrow, indicating a primary role for the hematopoietic system. When na€ıve T cells were skewed to Th2 cells, Hox5 mutant cells produced more Th2 cytokines and expressed more Gata3 than WT cells. Expression of HOX5 protein in Jurkat cells resulted in binding to the Gata3 gene at known STAT6 sites, indicating cooperation between these transcription factors in Gata3 gene regulation. CONCLUSIONS: These results indicate a novel role for HOX5 proteins as regulators of Th2 cell differentiation during allergic disease.
270
Whole Genome Sequencing Identifies Four Novel Variants in the Epidermal Differentiation Complex That Increase Risk and Severity for Atopic Dermatitis
Rasika A. Mathias1, Sameer Chavan2, Meher Boorgula3, William O. C. Cookson, MD, DPhil4, Saffron Willis-Owen5, Nicholas M. Rafaels6, Jon M. Hanifin, MD, FAAAAI7, Amy Paller8, Lynda C. Schneider, MD, FAAAAI9, Richard Gallo, MD, PhD10, Emma Guttman-Yassky, MD, PhD11, Peck Y. Ong, MD, FAAAAI12, Ingo Ruczinski13, Terri Beaty1, Li Gao1, Lisa A. Beck, MD, FAAAAI14, Miriam Moffat15, Donald Y. M. Leung, MD, PhD, FAAAAI16, and Kathleen C. Barnes, PhD, FAAAAI17; 1Johns Hopkins, BALTIMORE, 2University of Colorado, denver, 3University of Colorado, DENVER, 4Imperial College London, London, United Kingdom, 5Imperial College, LONDON, United Kingdom, 6Department of Medicine, University of Colorado, Denver, 7Oregon Health and Science University, Portland, OR, 8Northwestern University, CHICAGO, 9Harvard Medical School, Boston, MA, 10Division of Dermatology, University of California, San Diego, San Diego, CA, 11 Mount Sinai Health System - Dermatology, New York, NY, 12Children’s Hospital Los Angeles/USC, Los Angeles, CA, 13Johns Hopkins University, BALTIMORE, 14Department of Dermatology, University of Rochester Medical Center, Rochester, NY, 15Imperial College, LONDON, 16 K926i, National Jewish Health, Denver, CO, 17University of Colorado Denver, Aurora, CO. RATIONALE: Filaggrin (FLG) mutations located within the Epidermal Differentiation Complex (EDC; chr1:151972910-153642037) are known to increase risk for atopic dermatitis (AD). However, the role of variants within additional EDC genes is poorly understood. METHODS: We use whole genome sequencing on 493 European American AD subjects (average age 5 26, average severity measured by
EASI score 5 14.5) and 237 non-atopic controls in the Atopic Dermatitis Research Network (ADRN) study to test for association with single nucleotide polymorphisms (SNPs) in the EDC as risk determinants of AD as well as severity of disease. Replication was performed in an independent European sample of 609 AD cases and 695 controls. RESULTS: We identified 12 SNPs with a p <0.001 comparing AD to nonatopic controls including the well documented stop gain mutation in FLG (rs61816761, p 5 0.0003). Four of these SNPs were replicated in an independent European population (p<0.00001) including the stop gain mutation in FLG, two intergenic SNPs and one noncoding RNA (FLGAS1). Odds ratios for all SNPs were >3 in the ADRN dataset and ranged from 2.5-3 in the replication dataset. Severity of disease as measured by total serum IgE, eosinophil count, phadiatop and EASI score in all the AD subjects from the ADRN study increased with carrier status (i.e. AD subjects with all four risk alleles were more severe compared to those with 0-3 risk alleles, p<0.05). CONCLUSIONS: We have identified for the first time that non FLG variants in the EDC could result in added burden for risk and increased severity of AD.
271
A Cross-Sectional Study of Asthma Exacerbations in the Emergency Department: Viral Infections and the Microbiome
Joshua Kennedy, MD1,2, Olga Hardin, MD3, Jesse Denson, PhD4, Kurt Schwalm, BS5, Suzanne Godbold, RRT, AE-C3, Lee Crawley, MS, RRT-NPS3, Thomas Abramo, MD6, Ashley N. Stoner, MD3, John C. Kincaid, MD7, and Darrell L. Dinwiddie, PhD5; 1Arkansas Children’s Research Institute, Little Rock, AR, 2Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR, 3University of Arkansas for Medical Sciences, Little Rock, AR, 4University of New Mexico, Albuquerque, NM, 5Department of Pediatrics, University of New Mexico HSC, Albuquerque, NM, 6university of Arkansas for Medical Sciences, Little Rock, AR, 7University of Arkansas for Medical Sciencess, Little Rock, AR. RATIONALE: We employ next-generation sequencing to evaluate viral genomes and microbiomes of children with asthma exacerbations and controls in order to compare clinical markers of disease severity. METHODS: We enrolled children from the emergency department with asthma exacerbations and children with cold symptoms (controls). We obtained nasal pharyngeal swabs, Asthma Control Tests (ACT), and modified Jackson criteria (MJC). To identify viral infections, we used targeted RNA hybridization-based genome detection methods that included 34 respiratory viruses. We sequenced the variable regions 4-6 of 16S ribosomal RNA gene. Clinical data was compared between viralinduced asthma, asthma, viral-induced colds, and colds. Microbiome data was used to determine if specific bacteria or diversity were associated with viral-induced wheezing. RESULTS: ACT scores were lower in asthmatics with virus compared to asthmatics without virus (med517 vs. 20, respectively; p<0.05). Viralinduced asthmatics had worse lower respiratory symptoms by MJC than viral-induced colds control (med58 and 4, respectively; p<0.05). We identified five microbiome profiles classified by dominant genus, Moraxella, Corynebacterium, Staphylococcus, Haemophilus, and Diverse. Controls were Moraxella-dominant in 52.9% and Diverse in 17.6%. Asthmatics were 27.6% Moraxella-dominant and 37.9% Diverse. The microbiome of asthmatics with respiratory syncytial virus (RSV) infection trended towards a greater number of species than the microbiome from subjects with other viruses (mean 362 (RSV) vs. 276 (Other viruses); p50.055). CONCLUSIONS: Viral-induced asthma exacerbations are more likely to occur in children with less overall asthma control. Lower respiratory symptoms are more pronounced in asthmatics with viruses than in controls. Asthmatics tend to have a more diverse nasopharyngeal microbiota.
SATURDAY
Abstracts AB85
J ALLERGY CLIN IMMUNOL VOLUME 139, NUMBER 2