PLAUR polymorphisms are associated with asthma, PLAUR levels, and lung function decline  Sheila J. Barton, MSc, Gerard H. Koppelman, MD, PhD, Judith M. Vonk, PhD, Claudia A. Browning, BSc, Ilja M. Nolte, PhD, Ceri E. Stewart, PhD, Sue Bainbridge, BSc, Stacey Mutch, BSc, Matthew J. Rose-Zerilli, BSc, Dirkje S. Postma, MD, PhD, Nikolas Maniatis, PhD, Amanda P. Henry, PhD, Ian P. Hall, MD, DM, Stephen T. Holgate, MD, DSc, Patrick Tighe, PhD, John W. Holloway, PhD, Ian Sayers, PhD  Journal of Allergy and Clinical Immunology  Volume 123, Issue 6, Pages 1391-1400.e17 (June 2009) DOI: 10.1016/j.jaci.2009.03.014 Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Phase 1: microsatellite linkage and association analyses. A, Multipoint linkage analyses in the Nottingham families (n = 46 families). B, Association analyses in the Southampton families (n = 341 families). SPT, Skin prick test. Journal of Allergy and Clinical Immunology 2009 123, 1391-1400.e17DOI: (10.1016/j.jaci.2009.03.014) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Phase 2: Mapping of the D19S900-D19S178 interval using haplotype tagging SNPs (htSNPs). A, Haplotype block structure of D19S900-D19S178 (Chrs19:48845000-49100000). Seven Ensembl genes spanning the interval are shown: PLAUR, IRGC, LOC56006, KCNN4, LYPD5, ZNF283, and ZNF404. The intensity of shading represents D' (a measure of LD). B, LDU map of the same region,20 including the location of htSNPs (triangles). C, Single-point FBAT analysis in the Nottingham families. D, Single-point FBAT analysis in the Southampton families. Case (first affected sibling; n = 341) and control (n = 184) SNP analysis (triangles). Journal of Allergy and Clinical Immunology 2009 123, 1391-1400.e17DOI: (10.1016/j.jaci.2009.03.014) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Phase 2: Mapping of the D19S900-D19S178 interval using haplotype tagging SNPs (htSNPs). A, Haplotype block structure of D19S900-D19S178 (Chrs19:48845000-49100000). Seven Ensembl genes spanning the interval are shown: PLAUR, IRGC, LOC56006, KCNN4, LYPD5, ZNF283, and ZNF404. The intensity of shading represents D' (a measure of LD). B, LDU map of the same region,20 including the location of htSNPs (triangles). C, Single-point FBAT analysis in the Nottingham families. D, Single-point FBAT analysis in the Southampton families. Case (first affected sibling; n = 341) and control (n = 184) SNP analysis (triangles). Journal of Allergy and Clinical Immunology 2009 123, 1391-1400.e17DOI: (10.1016/j.jaci.2009.03.014) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 A, Schematic representation of the PLAUR gene showing the location of phase 3 SNPs. The PLAUR gene spans ∼40 kb on chromosome 19. Exons are depicted as open boxes except for alternatively spliced exon 7 (gray). B, Haplotype block structure of phase 3 PLAUR SNPs in the Southampton cohort (n = 1508 individuals). The intensity of shading represents D' (a measure of LD), and numerical values are given. Haplotype blocks were identified by using CIs. The location of SNPs showing association with asthma in this cohort are highlighted (∗P < .05, FBAT additive model). SNPs are labeled 1 to 18 and correspond to SNPs 1 to 18 in Table III. Journal of Allergy and Clinical Immunology 2009 123, 1391-1400.e17DOI: (10.1016/j.jaci.2009.03.014) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Phase 3: family-based association analyses of PLAUR SNPs with asthma and lung function phenotypes. Data represent summary P values obtained running FBAT using the biallelic additive model using data from the Southampton (n = 341 families; A), Nottingham (n = 46 families; B), and Dutch (n = 200 families; C) cohorts. P < .05 was considered significant (dashed line). Journal of Allergy and Clinical Immunology 2009 123, 1391-1400.e17DOI: (10.1016/j.jaci.2009.03.014) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Annual FEV1 decline in mL/y (with 95% CI) in subjects with asthma for the rs2356338 (5′ untranslated region [UTR] -649), rs4802189 (3′ UTR), and rs4803648 (3′ UTR) SNPs of PLAUR. Comparison of the common genotype groups to other genotype groups is shown. Data adjusted for height, sex, corticosteroid use, and smoking pack-years (see text). Median (inter-quartile range) number of readings per individual, 22.5 (6-38). Median (IQR) number of years per individual, 20.4 (10.9-28.2). Journal of Allergy and Clinical Immunology 2009 123, 1391-1400.e17DOI: (10.1016/j.jaci.2009.03.014) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

LD plots of association region using phase 2 haplotype tagging SNP data. A, Haplotype block structure generated from phase 2 htSNP (1-15) data in the Nottingham families (n = 46 families) using Haploview software. B, Haplotype block structure in the Southampton families (n = 341 families). The intensity of shading represents D'. ∗SNPs showing single-point association in the FBAT analyses. Journal of Allergy and Clinical Immunology 2009 123, 1391-1400.e17DOI: (10.1016/j.jaci.2009.03.014) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Expression profiling of genes and predicted genes in the phase 2 association region. A, Diagrammatic representation of the associated region in the single-point and haplotype analyses (Chromosome 19:48,841,000-48,938,000). ∗SNPs showing single-point association in the FBAT analyses. The region tagged by the 6 SNPs is shown spanning 33.8 kb and encompassing the 5′ UTR, exon I, and exon II of PLAUR. The locations of microsatellite markers D19S900 and D19S913 are shown. B, PCR amplification of PLAUR (exon 2-3), GeneScan 1 (PLAUR exon 1-predicted exon 1), and GeneScan2 (predicted exon 1-2 and 4-5) in various tissues and cell types. Markers of 100, 200, and 300 bp are shown. All assays were designed to generate a 100 to 300-bp transcript from cDNA and a 2 to 6-kbp transcript from genomic DNA as a positive control (data not shown). ASM, Airway smooth muscle; GeneScan1, predicted gene GeneScan00000009475; GeneScan2, predicted gene GeneScan00000030955; HBEC, human bronchial epithelial cell (U, undifferentiated; D, differentiated); IRGC, immunity-related guanosine triphosphatase (GTPase) family, cinema; PLAUR(1-3), splice variants 1, 2, 3E15,E16; THP-1, monocyte cell line. Journal of Allergy and Clinical Immunology 2009 123, 1391-1400.e17DOI: (10.1016/j.jaci.2009.03.014) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

The potential role of PLAUR in asthma The potential role of PLAUR in asthma. ECM, Extracellular matrix; ERK, extracellular signal-regulated kinase; FAK, Focal adhesion kinase; LO, lipoxygenase; MMP, matrix metalloproteinases; PI3K, phosphoinoside 3-kinase; Akt, protein kinase B; PLAU, urokinase plasminogen activator. Adapted from Crippa MP. Urokinase-type plasminogen activator. Int J Biochem Cell Biol 2007;39:690-4E17; and Kucharewicz I, Kowal K, Buczko W, Bodzenta-Lukaszyk A. The plasmin system in airway remodeling. Thromb Res 2003;112:1-7.E18 Journal of Allergy and Clinical Immunology 2009 123, 1391-1400.e17DOI: (10.1016/j.jaci.2009.03.014) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions