- Email: [email protected]
Filaggrin Associated Risk for Atopic Dermatitis Is Offset By Protective Missense Variants in Rptn and LCE1B Genes in the Epidermal Differentiation Complex
AB182 Abstracts
595
Measurement of Major Allergen Mus m 1 in Commercial Mouse Allergen Extracts and Mouse Urine
Taruna Khurana, PhD1, Jessica R. Shartouny2, Natalie A. David2, Jay E. Slater, MD1; 1FDA/CBER/OVRR/DBPAP, Silver Spring, MD, 2FDA/ CBER/OVRR/DBPAP. RATIONALE: Mus m 1 is a major allergen found in mouse urine and has been shown to contribute to asthma prevalence in inner city homes. Commercially available mouse epithelium extracts are non-standardized. Our goal is to develop monoclonal-antibody-based sandwich ELISA assays for measurement of Mus m 1. METHODS: Monoclonal antibodies were generated in rats by immunizing with natural Mus m 1 using a proprietary MonoExpress immunization protocol (GenScript USA). Anti-Mus m 1 polyclonal sera were generated in rabbits hyperimmunized with Mus m 1: rabbits were primed subcutaneously with 100 mcg of nMus m 1 in Freund’s Complete Adjuvant, followed by boosts of 100 mcg nMus m 1 in Freund’s Incomplete Adjuvant at weeks 3 and 7. Hybridoma supernatants and polyclonal sera were screened against nMus m 1 and mouse urine. Two sandwich ELISAs (sELISAs) were developed: one in which both the capture (12B10) and detection (biotinylated 11G6) antibodies were monoclonal, and one in which the detection antibody was rabbit polyclonal sera. RESULTS: The monoclonal assay was found to be highly specific and sensitive, and mouse urine contained 3.0 g/L of the antigen, but Mus m 1 in commercial extracts was below the limit of detection. Using polyclonal rabbit sera for detection, the assay was more sensitive. With this assay the Mus m 1 content of mouse urine was 6.7x10-1 g/L and the commercial extracts contained 1.5x10-4 to 2.5x10-3 g/L. CONCLUSIONS: sELISA assays has been developed for determining Mus m 1 contents of non-standardized mouse allergen extracts.
596
SUNDAY
Filaggrin Associated Risk for Atopic Dermatitis Is Offset By Protective Missense Variants in Rptn and LCE1B Genes in the Epidermal Differentiation Complex
Rasika A. Mathias, ScD1, Meher Boorgula2, Sameer Chavan, MS2, Kruthika R. Iyer2, Nicholas M. Rafaels2, Joseph Potee2, Jon M. Hanifin, MD, FAAAAI3, Amy S. Paller4, Lynda C. Schneider, MD, FAAAAI5, Richard L. Gallo, MD, PhD6, Emma Guttman-Yassky, MD, PhD7, Peck Y. Ong, MD, FAAAAI8, Ingo Ruczinski, PhD9, Terri H. Beaty, PhD9, Li Gao, MD, PhD2, Lisa A. Beck, MD, FAAAAI10, Donald Y. M. Leung, MD, PhD, FAAAAI11, Kathleen C. Barnes, PhD, FAAAAI2; 1Division of Allergy and Clinical Immunology, Demartment of Medicine, Johns Hopkins University, Baltimore, MD, 2Division of Allergy and Clinical Immunology, Department of Medicine, Johns Hopkins University, Baltimore, MD, 3Oregon Health and Science University, Portland, OR, 4 Northwestern University Feinberg School of Medicine, Chicago, IL, 5 Boston Children’s Hospital, Boston, MA, 6Division of Dermatology, University of California, San Diego, San Diego, CA, 7Icahn Medical School at the Mount Sinai Medical Center, New York, NY, 8Children’s Hospital Los Angeles/USC, Los Angeles, CA, 9Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, 10 Department of Dermatology, University of Rochester Medical Center, Rochester, NY, 11Department of Pediatrics, National Jewish Health, Denver, CO. RATIONALE: Null mutations in Filaggrin (FLG) located within the Epidermal Differentiation Complex (EDC; chr1:151972910-153642037) are known to increase risk for atopic dermatitis (AD); but the role of variants within additional genes in the EDC is not well understood. We implemented a whole genome sequencing (WGS) study to fully understand the genetic determinants of AD within the EDC. METHODS: Deep WGS (;30x) was performed on 493 European American AD subjects and 237 non-atopic controls. We assessed
J ALLERGY CLIN IMMUNOL FEBRUARY 2016
association with single nucleotide variants (SNVs) in the EDC, a region rich in genes important for epidermal maturation and examined these associations as a function of FLG carrier status (carrier defined as an individual with 1+ FLGrisk variants). RESULTS: We identified 17,231 SNVs; 14 SNVs had a p <0.001 comparing AD to non-atopic controls, including two functional missense variants. In contrast to the increased risk conferred by FLG variants _3), the newly identified missense variants in the genes encoding rep(OR> etin (RPTN; p50.0005, OR50.53) an extracellular epidermal matric protein, and late cornified envelope 1B (LCE1B, p50.0002, OR50.29) were both strongly protective. Importantly, the risk for AD in individuals that were carriers of FLG variants and RPTN or LCE1B variants was lower (OR52.36) as compared to carriers of FLGvariants only (OR5 3.4) supporting an overall protection conferred by these newly identified missense variants in the EDC. CONCLUSIONS: Relying on a sequencing approach, we identify genetic variants within the EDC that confer protection against AD. We demonstrate that these variants may offset the risk conferred by known variants in FLG.
597
The Role of Gastrin Releasing Peptide (GRP) in Atopic Dermatitis (AD) Induced By Interleukin 13 (IL-13)
Eun Byul Choi, Master Degree1, Zhou-Feng Chen, PhD2, Zhou Zhu, MD, PhD3, Tao Zheng, MD1; 1Yale University School of Medicine, New Haven, CT, 2st. LouisWashington University School of Medicine at, St Louis, MO, 3Yale University School of Medicine, CT. RATIONALE: The serum levels of GRP correlate with the human AD severity, and GRP has been implicated in itch in chemical-induced dermatitis. We previously showed selective expression of IL-13 in the skin caused a chronic pruritic AD. We investigated the role of GRP in a chronic AD model induced by IL-13. METHODS: GRP-deficient-IL-13-Tg-mice (IL-13Tg(+)/GRP-/-) was generated by crossbreeding GRP-/-knockout mice with K5-tTA-IL-13 mice (C576/BL/6 background). IL-13 is inducibly activated in the skin under keratin-5 (K5) promoter. The time to the onset of AD, AD Score, itch/ scratch behavior, dermal cytokines and serum IgE levels by ELISA were evaluated. Histopathological assessments (IHC, H&E, and Toluidine blue staining) of dermal infiltrate were performed. Expression of GRP by IF was determined using skin biopsy samples from AD patients and normal subjects. RESULTS: The expression of GRP was markedly enhanced in lesional skin of IL-13-induced AD and in dermal inflammatory cells and PGP9.5+ afferents in human AD skin, but not in the skin of control subjects. Compared to IL-13Tg(+)/GRP+/+ mice, IL-13Tg(+)/GRP–/–mice had significantly delayed onset of AD (P<0.01), attenuated itch-evoked scratching behaviors (P<0.01), reduced serum IgE levels (P<0.05), and substantially reduced dermal inflammatory responses. Deletion of GRP did not alter the transgene IL-13 activation. CONCLUSIONS: GRP is highly expressed in human AD skin and in IL13-induced AD skin. Deletion of GRP in IL-13 Tg mice markedly attenuated the AD phenotype, suggesting that GRP contributes to the onset of AD, generation of pruritus and Th2 immune response induced by IL-13.
595
Measurement of Major Allergen Mus m 1 in Commercial Mouse Allergen Extracts and Mouse Urine
Taruna Khurana, PhD1, Jessica R. Shartouny2, Natalie A. David2, Jay E. Slater, MD1; 1FDA/CBER/OVRR/DBPAP, Silver Spring, MD, 2FDA/ CBER/OVRR/DBPAP. RATIONALE: Mus m 1 is a major allergen found in mouse urine and has been shown to contribute to asthma prevalence in inner city homes. Commercially available mouse epithelium extracts are non-standardized. Our goal is to develop monoclonal-antibody-based sandwich ELISA assays for measurement of Mus m 1. METHODS: Monoclonal antibodies were generated in rats by immunizing with natural Mus m 1 using a proprietary MonoExpress immunization protocol (GenScript USA). Anti-Mus m 1 polyclonal sera were generated in rabbits hyperimmunized with Mus m 1: rabbits were primed subcutaneously with 100 mcg of nMus m 1 in Freund’s Complete Adjuvant, followed by boosts of 100 mcg nMus m 1 in Freund’s Incomplete Adjuvant at weeks 3 and 7. Hybridoma supernatants and polyclonal sera were screened against nMus m 1 and mouse urine. Two sandwich ELISAs (sELISAs) were developed: one in which both the capture (12B10) and detection (biotinylated 11G6) antibodies were monoclonal, and one in which the detection antibody was rabbit polyclonal sera. RESULTS: The monoclonal assay was found to be highly specific and sensitive, and mouse urine contained 3.0 g/L of the antigen, but Mus m 1 in commercial extracts was below the limit of detection. Using polyclonal rabbit sera for detection, the assay was more sensitive. With this assay the Mus m 1 content of mouse urine was 6.7x10-1 g/L and the commercial extracts contained 1.5x10-4 to 2.5x10-3 g/L. CONCLUSIONS: sELISA assays has been developed for determining Mus m 1 contents of non-standardized mouse allergen extracts.
596
SUNDAY
Filaggrin Associated Risk for Atopic Dermatitis Is Offset By Protective Missense Variants in Rptn and LCE1B Genes in the Epidermal Differentiation Complex
Rasika A. Mathias, ScD1, Meher Boorgula2, Sameer Chavan, MS2, Kruthika R. Iyer2, Nicholas M. Rafaels2, Joseph Potee2, Jon M. Hanifin, MD, FAAAAI3, Amy S. Paller4, Lynda C. Schneider, MD, FAAAAI5, Richard L. Gallo, MD, PhD6, Emma Guttman-Yassky, MD, PhD7, Peck Y. Ong, MD, FAAAAI8, Ingo Ruczinski, PhD9, Terri H. Beaty, PhD9, Li Gao, MD, PhD2, Lisa A. Beck, MD, FAAAAI10, Donald Y. M. Leung, MD, PhD, FAAAAI11, Kathleen C. Barnes, PhD, FAAAAI2; 1Division of Allergy and Clinical Immunology, Demartment of Medicine, Johns Hopkins University, Baltimore, MD, 2Division of Allergy and Clinical Immunology, Department of Medicine, Johns Hopkins University, Baltimore, MD, 3Oregon Health and Science University, Portland, OR, 4 Northwestern University Feinberg School of Medicine, Chicago, IL, 5 Boston Children’s Hospital, Boston, MA, 6Division of Dermatology, University of California, San Diego, San Diego, CA, 7Icahn Medical School at the Mount Sinai Medical Center, New York, NY, 8Children’s Hospital Los Angeles/USC, Los Angeles, CA, 9Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, 10 Department of Dermatology, University of Rochester Medical Center, Rochester, NY, 11Department of Pediatrics, National Jewish Health, Denver, CO. RATIONALE: Null mutations in Filaggrin (FLG) located within the Epidermal Differentiation Complex (EDC; chr1:151972910-153642037) are known to increase risk for atopic dermatitis (AD); but the role of variants within additional genes in the EDC is not well understood. We implemented a whole genome sequencing (WGS) study to fully understand the genetic determinants of AD within the EDC. METHODS: Deep WGS (;30x) was performed on 493 European American AD subjects and 237 non-atopic controls. We assessed
J ALLERGY CLIN IMMUNOL FEBRUARY 2016
association with single nucleotide variants (SNVs) in the EDC, a region rich in genes important for epidermal maturation and examined these associations as a function of FLG carrier status (carrier defined as an individual with 1+ FLGrisk variants). RESULTS: We identified 17,231 SNVs; 14 SNVs had a p <0.001 comparing AD to non-atopic controls, including two functional missense variants. In contrast to the increased risk conferred by FLG variants _3), the newly identified missense variants in the genes encoding rep(OR> etin (RPTN; p50.0005, OR50.53) an extracellular epidermal matric protein, and late cornified envelope 1B (LCE1B, p50.0002, OR50.29) were both strongly protective. Importantly, the risk for AD in individuals that were carriers of FLG variants and RPTN or LCE1B variants was lower (OR52.36) as compared to carriers of FLGvariants only (OR5 3.4) supporting an overall protection conferred by these newly identified missense variants in the EDC. CONCLUSIONS: Relying on a sequencing approach, we identify genetic variants within the EDC that confer protection against AD. We demonstrate that these variants may offset the risk conferred by known variants in FLG.
597
The Role of Gastrin Releasing Peptide (GRP) in Atopic Dermatitis (AD) Induced By Interleukin 13 (IL-13)
Eun Byul Choi, Master Degree1, Zhou-Feng Chen, PhD2, Zhou Zhu, MD, PhD3, Tao Zheng, MD1; 1Yale University School of Medicine, New Haven, CT, 2st. LouisWashington University School of Medicine at, St Louis, MO, 3Yale University School of Medicine, CT. RATIONALE: The serum levels of GRP correlate with the human AD severity, and GRP has been implicated in itch in chemical-induced dermatitis. We previously showed selective expression of IL-13 in the skin caused a chronic pruritic AD. We investigated the role of GRP in a chronic AD model induced by IL-13. METHODS: GRP-deficient-IL-13-Tg-mice (IL-13Tg(+)/GRP-/-) was generated by crossbreeding GRP-/-knockout mice with K5-tTA-IL-13 mice (C576/BL/6 background). IL-13 is inducibly activated in the skin under keratin-5 (K5) promoter. The time to the onset of AD, AD Score, itch/ scratch behavior, dermal cytokines and serum IgE levels by ELISA were evaluated. Histopathological assessments (IHC, H&E, and Toluidine blue staining) of dermal infiltrate were performed. Expression of GRP by IF was determined using skin biopsy samples from AD patients and normal subjects. RESULTS: The expression of GRP was markedly enhanced in lesional skin of IL-13-induced AD and in dermal inflammatory cells and PGP9.5+ afferents in human AD skin, but not in the skin of control subjects. Compared to IL-13Tg(+)/GRP+/+ mice, IL-13Tg(+)/GRP–/–mice had significantly delayed onset of AD (P<0.01), attenuated itch-evoked scratching behaviors (P<0.01), reduced serum IgE levels (P<0.05), and substantially reduced dermal inflammatory responses. Deletion of GRP did not alter the transgene IL-13 activation. CONCLUSIONS: GRP is highly expressed in human AD skin and in IL13-induced AD skin. Deletion of GRP in IL-13 Tg mice markedly attenuated the AD phenotype, suggesting that GRP contributes to the onset of AD, generation of pruritus and Th2 immune response induced by IL-13.