Published ahead of print on March 15, 2007, doi:10.1164/rccm.200607-1012OC
© 2007 American Thoracic Society doi: 10.1164/rccm.200607-1012OC
Association of Urokinase-type Plasminogen Activator with Asthma and Atopy1 University of Montreal Community Genomic Medicine Centre, Chicoutimi University Hospital, Saguenay, Quebec, Canada; 2 Department of Medicine, University of Montreal, Montreal, Quebec, Canada; 3 Department of Medicine, Université Laval, Laval, Quebec, Canada; 4 James Hogg iCAPTURE Centre, University of British Columbia, Vancouver, British Columbia, Canada; 5 McGill University and Genome Quebec Innovation Centre, Montreal, Quebec, Canada; 6 Department of Fundamental Sciences, Université du Québec à Chicoutimi, Saguenay, Quebec, Canada; 7 Department of Pediatrics and Child Health, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; 8 Faculty of Pharmacy and Department of Community Health Sciences, Manitoba Centre for Health Policy, Winnipeg, Manitoba, Canada; and 9 Ontario Institute for Cancer Research, Toronto, Ontario, Canada Correspondence and requests for reprints should be addressed to Catherine Laprise, Ph.D., Université du Québec à Chicoutimi, 555 Boulevard de l'Université, Chicoutimi, PQ, Canada G7H 2B1. E-mail: catherine_laprise{at}uqac.ca
Rationale: Urokinase plasminogen activator (uPA) interacts with its receptor on inflammatory and migrating cells to regulate extracellular matrix degradation, cell adhesion, and inflammatory cell activation. It is necessary for the development of an appropriate immune response and is involved in tissue remodeling. The PLAU gene codes for this enzyme, and is located on 10q24. This region has demonstrated evidence for linkage in a genome scan for asthma in a sample from northeastern Quebec. Here, we hypothesized that uPA may function as a regulator of asthma susceptibility. Objectives: To test for association between asthma and genetic variants of PLAU. Methods: We sequenced PLAU and tested for genetic association between identified variants and asthma-related traits in a French-Canadian familial collection (231 families, 1,139 subjects). Additional association studies were performed in two other family-based Canadian cohorts (Canadian Asthma Primary Prevention Study [CAPPS], 238 trios; and Study of Asthma Genes and the Environment [SAGE], 237 trios). Measurements and Main Results: In the original sample, under the dominant model, the common alleles, rs2227564C (P141) and rs2227566T, were associated with asthma (p = 0.011 and 0.045, respectively) and with airway hyperresponsiveness (AHR) (p = 0.026 and 0.038, respectively). Analysis of the linkage disequilibrium pattern also revealed association of the common haplotype for asthma, atopy, and AHR (p = 0.031, 0.043, and 0.006, respectively). Whereas no significant association was detected for PLAU single-nucleotide polymorphisms in the CAPPS cohort, association was observed in the SAGE cohort between the rs4065C allele and atopy under additive (p = 0.005) and dominant (p = 0.0001) genetic models. Conclusions: This suggests a role for the uPA pathway in the pathogenesis of the disease.
Key Words: airway hyperresponsiveness • association study • asthma • atopy • haplotypes • urokinase-type plasminogen activator gene
Asthma is recognized as a complex trait, and the genetic risk factors for its development, progression, and subphenotypes remain largely unknown despite several genetic studies performed in the past decade. In a genome scan conducted in 2004 to identify susceptibility loci for asthma and related phenotypes in a French-Canadian population, eight chromosomal regions with suggestive evidence for linkage were identified (1). Two regions were suggestive for asthma (6q26 and 16p12.1) and two for atopy (10q24 and 11q13) phenotypes, whereas the remaining four were associated with elevated IgE levels (5q23.3, 6q27, 7q22.2, and 9q21.32). This was the first time that the 10q24 region was identified in a linkage study for asthma (logarithm of the odds = 2.32; p = 0.00016). In addition, this novel region was found to be associated with severe asthma in a more recent genome scan study conducted in a Puerto Rican population (p < 10–4) (2). The 10q24 region harbors the urokinase-type plasminogen activator (PLAU) gene, which is expressed in most cell types and encodes for the urokinase plasminogen activator (uPA), a secreted proteolytic enzyme involved in many biological processes, including inflammation and tissue remodeling (3). This enzyme is recruited to the cell membrane by the uPA receptor, where it interacts with both extracellular and membrane components (4) to activate pericellular plasmin-mediated proteolysis (3) and to activate several intracellular signaling pathways. It has notably been implicated in the activation and infiltration of lung tissue by T cells and eosinophils (5, 6). PLAU and its receptor have been shown to be up-regulated in a human cell culture model of asthma after application of compressive stress-mimicking bronchospasm, as well as in postmortem lung tissue from patients that died of status asthmaticus (7). Its two main inhibitors, SERPINE1 and SERPINB2, have also been shown to be overexpressed in asthmatic bronchial tissue (8), and a variant of SERPINE1 has been associated with an increased risk for asthma (9). To the best of our knowledge, the PLAU gene itself has never been studied for association with asthma. It has, however, been found to be associated with other diseases involving inflammation or extracellular proteolysis. The common PLAU gene variant, P141 (rs2227564C), has been associated with increased susceptibility for late-onset Alzheimer's disease (10, 11) and colorectal cancer invasion (12), whereas common 3' untranslated region (UTR) variant rs4065C has been associated with a decreased risk for rheumatoid arthritis (13), mitral valve prolapse (14), urolithiasis (15), and Alzheimer's disease (16). Combining positional cloning and candidate gene approaches, and based on reported genetic associations with other diseases, we hypothesized that PLAU may act as an asthma susceptibility gene. To verify this, we sequenced the PLAU gene and conducted an association study in a French-Canadian, family-based sample. We then investigated two additional Canadian trio-based cohorts for replication purposes. Some of the results of this study have been previously reported in the form of an abstract (17).
Samples The original study sample comprises 231 asthmatic families from Saguenay–Lac-St.-Jean (SLSJ) (1,139 subjects), a region of northeastern Quebec, Canada, recognized as a young founder population (18–22). This sample was described in a recent report (23), and all information concerning recruitment and clinical evaluation of subjects is available in the online supplement. Clinical characteristics are presented in Table 1. Briefly, asthma phenotype was characterized by the investigators for all participants using a respiratory health questionnaire and function tests following American Thoracic Society standards (22). Airway hyperresponsiveness (AHR) was defined as a positive challenge to methacholine (provocative concentration causing a 20% drop in FEV1 [PC20] < 8 mg/ml) at time of recruitment. If PC20 was not measurable, a 15% augmentation in FEV1 after inhalation of a bronchodilatator or a variation in PEF of at least 12% within a 2-week period was also considered diagnostic of AHR (22). Participants were defined as having asthma if (1) they had a reported history of asthma (validated by a physician), or (2) they showed asthma-related symptoms and a positive PC20 at the time of recruitment. Subjects were deemed atopic if they had at least one positive response (wheal diameter  3 mm at 10 min) on skin prick test (24). Subjects with a PC20 greater than 8 mg/ml; without history of physician-diagnosed asthma, and without both symptoms of asthma and a PC20 greater than 8 mg/ml; and with no positive response on skin prick test were considered unaffected for AHR, asthma, and atopy, respectively.
The Canadian Asthma Primary Prevention Study (CAPPS) cohort (25–27) and the Study of Asthma Genes and the Environment (SAGE) birth cohort were used as replication cohorts. These two cohorts are fully described in the online supplement, and clinical characteristics are presented in Table 2. Briefly, children affected with asthma or related phenotypes were selected along with their parents from these two population asthma cohorts to form study trios for replication analysis. Phenotypes were characterized for all children in each recruiting center, either at time of recruitment (for the SAGE cohort) or at 7 years of age (for the CAPPS cohort), and asthma was diagnosed by a pediatric allergist based on clinical evaluation. Challenge to methacholine was performed for all children in both cohorts. AHR was defined as a PC20 less than 6.6 mg/ml (28, 29). Atopy was defined as at least one positive response on skin prick test (24).
PLAU Sequencing To identify single-nucleotide polymorphisms (SNPs) for genetic analyses, 50 unrelated probands with asthma (25 males and 25 females) were selected from the SLSJ sample (validated using BALSAC population register [19]). PLAU sequence information was obtained from Ensembl version 33 (http://www.ensembl.org/index.html). The sequenced region spanned 7.6 kb, starting from 850 bp upstream of the mRNA transcription start site (30) to 430 bp downstream of the 3'UTR. The gene was divided in to 10 regions; the amplification primers, conditions, and material are presented in the online supplement. SNPs described in this article are cited using their reference sequence number (rs#) from the National Center for Biotechnology Information (NCBI) database, if documented.
Genotyping
Statistical Analysis Allele distribution patterns and Mendelian errors under the original sample were assessed by FBAT software (version 1.7) (32–34) in additive and dominant genetic models using the empirical variance estimator "-e" for each PLAU SNP and tested phenotypes (32). Hardy-Weinberg equilibrium (HWE) for each SNP and strength of linkage disequilibrium between pairs of SNPs, measured as D prime (D') (35), were done using Haploview software (version 3.31; Broad Institute of MIT and Harvard University, Boston, MA) (36). Regions of strongly associated markers were inferred using the definition proposed by Gabriel and colleagues (37) implemented in Haploview. Based on the linkage disequilibrium block of variants, haplotypes were inferred and assessed for nonrandom transmission using the "hbat" command of the FBAT software (Harvard University, Boston, MA) (38) under additive and dominant models using the empirical variance estimator "-e" (32). A similar approach was taken for the replication analyses in the CAPPS and SAGE cohorts. Indeed, the theoretical expression of the variance of the FBAT statistic was used (default parameters) under additive and dominant models. Haplotypes were inferred and assessed for both cohorts using the "hbat" command of the FBAT software under additive and dominant models.
PLAU Sequencing All exonic regions were sequenced, except for short gaps in exon 4 and in the 3'UTR; most of the intronic regions were also sequenced (Figure 1). As no documented SNPs are known in these gaps, and as we judged that we had sufficient SNP coverage, we did not complete the sequence of these gaps. Sequence analysis allowed the identification of 13 polymorphisms. The SNP selection criteria for the association study were based on the allele frequency, which had to be greater than 5% as determined by sequencing, and on the location within the PLAU gene, with priority placed on coding regions, regulatory regions (i.e., 5' and 3'UTR), and intron/exon junctions.
Seven SNPs were initially selected for PLAU characterization and association testing (presented in Figure 1 in the 5'  3' order): rs1916341, rs2227562, rs2227564, rs2227566, rs2227568, rs2227571, and rs4065. Among the SNPs identified, an A/T substitution located upstream of the PLAU gene was not documented in the NCBI SNP database (dbSNP). This SNP had an allele frequency less than 5% and, therefore, was not selected for the association study. Of the remaining five discarded SNPs, four were uninformative, with minor allele frequencies less than 0.025, and one SNP, rs2227560, had a failed genotyping assay. Among the seven selected SNPs were the two previously studied variants, rs2227564 (P141L) (10, 12) and rs4065 (13–15). In addition to this initial selection, SNP rs2688607 was genotyped in all three studies through the AllerGen (NCE, Inc.; www.allergen-nce.ca) gene panel (refer to the online supplement for details on its selection). It is located 7 kb upstream of exon 1 (SNP characteristics presented in Table 3).
Genotyping In both replication cohorts, genotyping completion rate was greater than 95% for the four SNPs, and there were no Mendelian errors in either the CAPPS or SAGE cohorts. We examined the evidence for departure from HWE separately for the parents and offspring. Significant deviation from HWE is observed in the parental generation of the SAGE cohort for rs4065 (p = 0.0031), and in the parental generation of the CAPPS cohort for rs2688607 (p = 0.006) and rs2227564 (p = 0.027). There was no evidence of departure from HWE in the offspring generation of either cohort. We note that one potential explanation for this finding may be admixture in the parental generation, as both the CAPPS and SAGE cohorts include minority populations. To examine this hypothesis, we stratified the cohorts by ethnicity and examined allele frequencies and HWE. We found that, for SNPs rs2688607 and rs4065, the major and minor allele depends upon population ethnicity, as the most frequent allele in the white population is the minor allele in the Asian population of these samples (see Table E3 in the online supplement). When stratified by ethnicity, the parental generation of the CAPPS cohort is in HWE for the Asian population, and there is only minor evidence of departure for rs2227564 (p = 0.04) in the white population. For the SAGE cohort, when testing for HWE stratified by ethnicity, there is no evidence for departure from HWE in any ethnicity. This suggests that the departure from HWE observed in the combined analyses of these cohorts is due to population stratification. One of the benefits of the trio design is protection against type 1 error due to population stratification. However, our data indicate that caution should be exercised when examining this locus in case-control designs.
Family-based Association Analyses of PLAU SNPs in the SLSJ Familial Sample
PLAU Linkage Disequilibrium and Haplotype-specific Association Analyses Figure 2 shows a Haploview linkage disequilibrium plot forming a unique block, indicating that SNPs are likely to be transmitted together. Within this region of high linkage disequilibrium, three haplotypes, H8 (1), H8 (2), and H8 (3), with frequencies higher than 5%, were observed (Table 6) and assessed for nonrandom transmission in the SLSJ familial sample (Table 7). Frequent haplotype H8 (1) was shown to be overtransmitted to probands with asthma, atopy, and AHR under the additive model (p = 0.024, 0.056, and 0.015, respectively) and to those with asthma, atopy, and AHR under the dominant genetic model (p = 0.031, p = 0.043, and p = 0.006, respectively).
Family-based Association Analyses of PLAU SNPs in the CAPPS and SAGE Cohorts Four PLAU SNPs were tested individually for association with asthma, atopy, and AHR under additive and dominant genetic models. No significant findings were found in the CAPPS cohort under either genetic model (see Tables E4 and E5). Analyses were, however, suggestive of a negative association between the rs2227562A and rs4065C alleles and AHR (p values between 0.06 and 0.12). In the SAGE cohort, a significant association between atopy and the rs4065C allele was found. Under the additive model (Table 8), the rs4065C allele was undertransmitted to children with atopy (p value = 0.005), and a similar pattern was observed under the dominant genetic model, with the rs4065C allele being undertransmited (p value = 0.0001) (Table 9). No association with asthma or AHR was observed in the SAGE cohort.
Haplotype analyses were also performed with the four PLAU SNPs (Haploview linkage disequilibrium plot presented in Figure E1). In the CAPPS cohort, the common H4 (1) haplotype was overtransmitted to children with AHR (p = 0.061, additive model), whereas the minor H4 (3) haplotype was significantly undertransmitted to the same children (p = 0.051 and 0.045, additive and dominant models, respectively) (Table E6). In the SAGE cohort, the common H4 (1) haplotype was overtransmitted to children with atopy (p = 0.010, additive) (see Table E7). These finding are consistent with the H8 haplotype analysis from the original SLSJ familial sample (Table 7). When using the same four SNPs for comparison, however, common haplotype H4 (1) fails to reach significance in the SLSJ sample (Table E8). This suggests that the additional SNPs are necessary to better delineate the functional variant in the original SLSJ sample. Given their similar structures, the CAPPS and SAGE samples were combined for further analyses (Tables E9 and E10). The results showed a negative association between allele rs2227562A and asthma (p = 0.046, additive; and p = 0.028, dominant), as well as between allele rs4065C and both asthma and AHR (p = 0.058 and p = 0.0002, dominant). Haplotype analysis showed common haplotype H4 (1) to be overtransmitted to probands with atopy (p = 0.006, additive), whereas minor haplotype H4 (3) was undertransmitted to probands with asthma and AHR (p = 0.049 and p = 0.050, dominant).
Using family-based samples, we observed associations between allelic variants of novel candidate gene PLAU and asthma phenotypes in three independent Canadian population samples. In the original French-Canadian familial sample, the common alleles of three SNPs, rs2688607, rs2227564C (P141), and rs2227566T, were associated with asthma and AHR. Additional studies were performed in two other Canadian cohorts (CAPPS and SAGE), in which common allele rs4065C exhibited a highly significant association with protection from atopy. These single-marker association results were further supported by haplotype association analyses, which identified significant, nonrandom distribution of marker haplotypes covering 12 kb of the PLAU gene. These results are in accord with current literature suggesting a role for the PLAU in asthma pathogenesis. At the cellular level, it is believed to increase pericellular proteolysis, which, in turn, releases chemotactic mediators and favors adhesion and migration of eosinophils, fibroblasts, and smooth muscle cells through the extracellular matrix in lung tissue (3–5, 39, 40). By activating its membrane receptor, it also triggers several intracellular pathways (39, 41), which induce rearrangement of cytoskeleton and cell movement (40, 41), as well as activation of inflammatory cells (42) and T-cell priming (43). Knockout mice for the PLAU gene fail to generate appropriate response to Cryptococcus neoformans infection and to schistosomal antigen challenge, suggesting that the uPA is essential for the development of both type 1 and type 2 immune responses (44, 45). Previous studies have also shown the rs2227564C (P141) and rs4065C alleles to be respectively associated with susceptibility and protection for other diseases involving inflammation and tissue remodeling (10–15). However, only the rs2227564 (P141L) variant has been studied for its functional impact. This C/T mutation codes for the nonsynonymous change of a proline (P) for a leucine (L) at amino acid position 141, located in the kringle domain of the urokinase protein (46). The kringle structure has the affinity for heparin, plasminogen, and extracellular matrix components, such as proteoglycans, and is necessary for binding to the urokinase receptor (12, 47). The L141 allele has been shown to decrease the affinity for fibrin, and possibly for other extracellular matrix components, by enhancing the hydrophobicity of the kringle structure (48). Little documentation is available for the other associated variants. As SNPs rs2688607 and rs2227566 fall in the same haplotype block as rs2227564 (P141L) (D' = 0.92 and 0.94, respectively), it is likely that these SNPs act as markers of P141L rather than as causal mutations. The rs4065 variant is located in the 3'UTR of PLAU mRNA, which contains multiple instability-determining regions (49). Although its functional impact has not been studied, it could be hypothesized that the rs4065 variant would modulate PLAU gene expression at a post-transcriptional level, either by directly modifying the stability of the 3'UTR or by altering its affinity for mRNA-stabilizing factors (50). It is also possible that rs4065 would be linked to another functional SNP located in a portion of 3'UTR not covered by gene sequencing. More functional work is needed to delineate the real impact of these SNPs in asthma pathogenesis. Populations with founder effect, such as the one from Saguenay–Lac-St.-Jean, are important in multigenic disease mapping because they exhibit a lower number of risk genes for a given disease, and thus allow the identification of susceptibility loci that would have otherwise been concealed by other high-risk loci (51). Neale and Sham have suggested that the gene, rather than specific variants or haplotypes, should be considered as the unit of replication (52). In a recent review, Ober and Hoffjan support this definition of replication, citing the many recent examples of established associations with different functional variants within the same gene in different populations (53). Therefore, although association results were not directly replicated in the SAGE cohort, the association of the rs4065C variant with protection from atopy does support the association of PLAU with asthma phenotypes, and provides evidence that it is not a unique feature of SLSJ. More work will however be needed to better judge the association's importance among, and interaction with, other genetic and environmental factors. In conclusion, we found that common alleles, rs2227564C (P141) and rs2227566C, were associated with asthma and AHR in a northeastern Quebec sample. We also found a common PLAU haplotype to be associated with susceptibility to asthma, AHR, and atopy. We have shown that these findings were consistent with data from two other Canadian cohorts, in which allele rs4065C could be associated with protection from atopy. This further supports the potential role of the plasminogen activating pathway in the pathogenesis of asthma.
The authors thank all families for their enthusiastic participation in this study. The authors also thank Diane Gagné, Muriel Grenon, and Dr. Paul Bégin for their invaluable participation in acquiring the subjects.
Supported by the Canadian Institutes of Health Research and the Respiratory Health Network of the Fonds de la Recherche en Santé du Québec, and by AllerGen NCE, Inc., which paid for the genotyping. P.B. and K.T. are supported by the Allergy, Genes and Environment Network (AllerGen). T.J.H. is recipient of an Investigator Award from the Canadian Institutes of Health Research and a Clinician–Scientist Award in Translational Research from the Burroughs Wellcome Fund. D.D. is supported by grants from the Canadian Institutes of Health Research, Institutes of Gender and Health, Genetics, Population and Public Health, and the IMPACT fellowship, and is also supported by the Lung Association of British Columbia. C.L. is the chair-holder of the Canada Research Chair on genetic determinants in asthma and the director of the Genetics platform of the Respiratory Health Network of the Fonds de la Recherche en Santé du Québec, which financially supports the Saguenay–Lac-St.-Jean sample. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200607-1012OC on March 15, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form July 24, 2006; accepted in final form March 7, 2007
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