Published ahead of print on August 24, 2006, doi:10.1164/rccm.200601-081OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200601-081OC
Polymorphisms in the Muscarinic Receptor 1 Gene Confer Susceptibility to Asthma in Japanese SubjectsFirst Department of Medicine and Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Correspondence and requests for reprints should be addressed to Nobuyuki Hizawa, M.D., First Department of Medicine, Hokkaido University Graduate School of Medicine, N-15 W-7, Kita-Ku, Sapporo 060-8638, Japan. E-mail: nhizawa{at}med.hokudai.ac.jp
Rationale: The human cholinergic receptor muscarinic-1 (CHRM1) is widely distributed in the lungs. In patients with asthma, CHRM1 may be involved in airway constriction, airway epithelial cell proliferation, and airway inflammation. The CHRM1 gene is located on chromosome 11q13, which is one of the candidate loci for asthma and atopy. Objectives: To determine the role of the CHRM1 gene polymorphisms in asthma. Methods: We studied nine single-nucleotide polymorphisms (–18379G > A, –9697C > T, –6965T > C, –4953A > G, +267A > C, +1353C > T, +3970C > G, +5418C > G, and +5455G > T) in a case-control study using 326 patients with asthma and 333 healthy control subjects. We also examined functional consequences of the –9697C > T and –4953A > G polymorphisms at the regulatory region using an mRNA reporter assay. Measurements and Main Results: Two common single-nucleotide polymorphisms (–9697C > T and –4953A > G) were associated with asthma. The odds ratio for the TT homozygotes at the –9697C > T polymorphism was 0.29 compared with the CC homozygotes (95% confidence interval, 0.12–0.73; p = 0.008), and the odds ratio for the GG homozygotes at the –4953A > G polymorphism was 1.86 compared with the AA homozygotes (95% confidence interval, 1.04–3.34; p = 0.038). Haplotype analysis showed that the –9697T/–6965T/–4953A haplotype was associated with a lower risk of asthma (p = 0.00055) and the –9697C/–6965T/–4953G haplotype was associated with an increased risk of asthma (p = 0.020). The –9697T/–4953A haplotype was also associated with lower luciferase activity in vitro compared with the –9697C/–4953G haplotype. Conclusions: This study, together with an in vitro functional study, suggests that the CHRM1 gene is an important susceptibility locus for asthma on chromosome11q13.
Key Words: case-control studies • IgE • muscarinic cholinergic receptor-1 • single-nucleotide polymorphism
The cholinergic nerves are the dominant neural bronchoconstrictor pathway in humans (1). They release acetylcholine onto muscarinic receptors causing cholinergic bronchoconstriction (2), mucous hypersecretion, and edema in the airways. Increases in cholinergic nerve activity and cholinergic hypersensitivity are associated with asthma, and patients with asthma are hypersensitive to the bronchoconstricting actions of muscarinic agonists (3). The human cholinergic receptor muscarinic 1 (CHRM1; Online Mendelian Inheritance of Man database no. 118510) is widely localized in the human lung, including the alveolar walls, bronchial epithelial cells, parasympathetic ganglia, neuromuscular junction, and submucosal glands (4). Studies using pirenzepine, a muscarinic antagonist selective for M1 receptors, have shown that M1 muscarinic receptors are involved in vagally induced bronchoconstriction (5–7). M1 receptor–deficient mice showed increased bronchoconstriction in response to 10–8 M muscarine in peripheral airways (8), suggesting the existence of an M1 receptor–dependent pathway counteracting cholinergic bronchoconstriction. M1 receptors also play a role in mast cell function (9), epithelial cell proliferation in the trachea (10), release of neutrophil and monocyte chemotactic activity from epithelial cells (11), acetylcholine-induced relaxation of the human pulmonary veins (12), and regulation of water and electrolyte secretion on submucosal glands (13). Taken together, CHRM1 is critically involved in the pathophysiology of asthma. The gene encoding CHRM1 exists on chromosome 11q13, which has been linked to asthma and asthma-related phenotypes in several genomewide searches (14–17). Given the important role of muscarinic cholinergic mechanisms in asthma, the CHRM1 gene is biologically an excellent candidate for asthma susceptibility in the region of chromosome 11q13. Thus, in the current study, we examined whether genetic variations in the CHRM1 gene are associated with asthma. To gain insight into the possible molecular basis of the disease association, we also examined functional consequences of single-nucleotide polymorphisms (SNPs) at the regulatory region of the CHRM1 gene.
See online supplement for additional details.
Study Subjects
Identification of Polymorphisms Genomic DNA from Japanese subjects was genotyped for +267A > C (rs2067477) and +1353C > T (rs2067480), because an association of these polymorphisms at the muscarinic M1 receptor gene with cognitive function in schizophrenic patients has been reported (19). We selected an additional seven SNPs for genotyping based on the frequency and location of SNPs and the linkage disequilibrium (LD) structure in and around the CHRM1 gene. We initially obtained genotyping data of 26 HapMap SNPs (spanning 31.6 kb around the gene) from 45 unrelated Japanese subjects at the International HapMap Project (available online at http://hapmap.org/). To select tagSNPs in this region, we used the multimarker predictor method implemented in the Tagger program (20). Tag set was generated (using a threshold r2 of 0.8) using 14 common SNPs with a minor allele frequency of more than 0.05 in the Japanese population. As the +267A > C and +1353C > T polymorphisms were in a complete LD, we genotyped a total of eight SNPs (–18379G > A [rs1938677], –9697C > T [rs2075748], –6965T > C [rs542269], –4953A > G [rs1942499], +1353C > T [rs2067480], 3970C > G [rs4963323], 5418C > G [rs11601597], and 5455G > T [rs11605665]) for all individuals (n = 659). These SNPs were typed using the assay that combines kinetic (real-time quantitative) polymerase chain reaction (PCR) with allele-specific amplification, as described elsewhere (18). The PCR products were detected using the ABI 7700 Sequence Detection System with the dsDNA-specific fluorescent dye SYBR Green I (Applied Biosystems, Foster City, CA). The –4953A > G polymorphism was typed using TaqMan assay (Applied Biosystems).
Statistical Analysis
Luciferase Reporter Gene Assay
Electrophoretic Mobility Shift Assay
Characteristics of the 333 healthy control subjects and 326 subjects with asthma are shown in Table 1. The median age of subjects with asthma was significantly higher than in healthy control subjects (p < 0.0001). There were significantly more females in the asthma group than in the control group (p < 0.0001). Subjects with asthma were more likely to be atopic and had higher levels of total serum IgE than did healthy control subjects (  2 test or analysis of variance, p < 0.0001). More than 50% of the control subjects were atopic, which is consistent with recent findings that the prevalence of atopy (as indicated by specific IgE against common inhaled allergens) among Japanese subjects is increasing (23, 24). Prebronchodilator baseline FEV1 at an initial visit to our hospital was examined for 293 subjects with asthma (89.9%), and improvement of FEV1 after bronchodilator therapy (400 µg salbutamol) or after a course of standard asthma medications (inhaled corticosteroids, long-acting  2-agonists, theophylline, or leukotriene modulators) was recorded for 214 (65.6%) subjects with asthma (Table 1). All eight of the SNPs investigated were within the HWE in the control group (p > 0.05). The overall success rate for genotyping was 99.6%. Of the eight SNPs, two common SNPs (–9697C > T [rs2075748] and –4953A > G [rs1942499]) in the regulatory region of the CHRM1 gene had a significant association with asthma (Table 2). Both of these SNPs were significantly associated with asthma when the analysis was adjusted for age, sex, smoking status, and atopic status (Table 3). The OR for the TT homozygotes of the –9697C > T polymorphism was 0.29 compared with the CC homozygotes (95% CI, 0.12–0.73; p = 0.008), and the OR for the GG homozygotes of the –4953A > G polymorphism was 1.86 compared with the AA homozygotes (95% CI, 1.04–3.34; p = 0.038).
We analyzed data from the eight SNPs with the Haploview program (25) and identified two haplotype blocks (Figure 1) in our case-control population. Haplotype block I comprised three SNPs in the regulatory region (–9697C > T [rs2075748], –6965T > C [rs542269], –4953A > G [rs1942499]), and haplotype block II comprised three SNPs (+1353C > T [rs2067480], +3970C > G [rs4963323], +5418C > G [rs11601597]) in the coding exon and the 3'-UTR. Haplotype analyses were performed in both blocks I and II. The frequency of CHRM1 haplotypes is shown in Table 4. In block I, the –9697T/–6965T/–4953A haplotype was associated with a significantly lower risk of asthma (p = 0.00055) and the –9697C/–6965T/–4953G haplotype was associated with a significantly increased risk of asthma (p = 0.020). Inspection of specific haplotypes revealed that this association is most likely due to –9697C > T and –4953A > G, because the same allele for –6965T > C is part of both risk and protective haplotypes. In contrast, none of the haplotypes in block II was associated with asthma.
In the case-only study, associations between asthma-related phenotypes, such as total serum IgE levels and atopy, and the polymorphisms of CHRM1 were also investigated. We could not find any significant association between the genotypes of the eight SNPs and total serum IgE levels or atopy (see Tables E1 and E2 in the online supplement). The transcriptional activity of the CHRM1 SNPs at the regulatory region was compared between the –9697C/–4953G haplotype and the –9697T/–4953A haplotype transiently transfected into human neuroblastoma IMR32 cells. Luciferase activity in cell extracts was assessed 24 h after transfection, and was expressed as fold increase in the activity of the CHRM1 reporter constructs compared with the pRL-TK vector. Figure 2 shows that the reporter plasmid carrying the –9697T/–4953A promoter displayed 37% lower transcriptional activity compared with the plasmid carrying the –9697C/–4953G promoter (p = 0.019). This difference in transcriptional activity was consistent in eight independent experiments.
EMSA failed to show a robust difference in binding affinity of NF-  B to the –9697T or the –9697C allele, or in binding affinity of USF-1 to the –4953A or the –4953G allele (data not shown).
Given a high a priori biological plausibility for asthma, we tested the hypothesis that the allelic variants at the regulatory region of the CHRM1 confer susceptibility to asthma by conducting a case-control study in a relatively large population of unrelated Japanese subjects. In accordance with our primary hypothesis, we found that the presence of the –9697CC genotype, –4953GG genotype, or the –9697C/–4953G haplotype at the regulatory region was significantly associated with a diagnosis of asthma. Our genetic association study had several strengths: first, muscarinic receptors, including M1, have been biologically implicated in the pathogenesis of asthma; second, the gene encoding the CHRM1 is located on chromosome 11q13, a genomic region linked to the diagnosis of asthma and atopy in several genome-wide scans (14–17); third, in vitro functional analyses have shown that the haplotype at the regulatory region has an effect on a basal promoter function in IMR32 cells, with the –9697T/–4953A haplotype associated with 37% decreased promoter activity compared with the –9697C/–4953G haplotype. Accordingly, our data suggest that the –9697/–4953 haplotype may influence the affinity of a particular nuclear protein to the regulatory region of the CHRM1 gene, resulting in altered transcriptional activity and ultimately leading to a higher or lower risk of asthma. Although the exact mechanisms underlying the involvement of the CHRM1 gene in the pathogenesis of asthma remain to be identified, several reports indicate that the cholinergic pathway has an important role in the pathogenesis of asthma, in particular in the regulation of bronchoconstriction, airway inflammation, and airway remodeling. An M1 receptor–dependent pathway counteracts cholinergic bronchoconstriction, possibly via the release of a relaxing agent (8); both respiratory epithelia and sympathetic nerve terminals within bronchial smooth muscle are equipped with M1 receptors (7, 26) and releasable bronchodilating agents, such as nitric oxide and prostaglandin E2 (27). Studies with the M1 receptor–preferring antagonist, pirenzepine, have also suggested the existence of pulmonary M1 receptors modulating airway diameter (28). Furthermore, Jones and colleagues (29) demonstrated that stable expression in RBL-2H3 mast cells of the M1 muscarinic acetylcholine receptor leads to carbachol-stimulated mast cell degranulation. An animal model of asthma showed that anticholinergic agents protect against allergen-induced airway remodeling (30). Together with these in vivo and in vitro findings, our findings support the contention that CHRM1 plays an important role in the pathogenesis of asthma. Our findings may be of considerable relevance to asthma treatment, providing an important basis for identification of individuals for whom the cholinergic pathway could be targeted.
Sequence analysis indicated that the T allele at the –9697C > T polymorphism creates a potential NF- In conclusion, given the important role of muscarinic cholinergic mechanisms in pulmonary disease, this case–control study, together with an in vitro functional analysis, suggests that the CHRM1 gene is an important susceptibility locus for asthma at chromosome 11q13. The –9697/–4953 haplotype at the regulatory region of the gene may contribute to the development of asthma by altering the human lung muscarinic receptor system in ways that could account for the increased in vivo lung cholinergic hyperresponsiveness found in patients with asthma.
The authors thank all of the subjects of this study for their participation. They also thank Yoshiko Obata and Tomoko Akiyama for their excellent technical assistance, and Takeshi Sawazaki at the Pharmaceutical Research Laboratory, Hitachi Chemical Co., Ltd., for kindly measuring Ag-specific IgE antibody levels (MAST).
Supported by the Japan Society for the Promotion of Science (grant 17590771). 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.200601-081OC on August 24, 2006 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 January 19, 2006; accepted in final form August 24, 2006
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