Published ahead of print on November 21, 2008, doi:10.1164/rccm.200809-1436OC
© 2009 American Thoracic Society doi: 10.1164/rccm.200809-1436OC
Chromosome 17q21 Gene Variants Are Associated with Asthma and Exacerbations but Not Atopy in Early Childhood1 Copenhagen Prospective Studies on Asthma in Childhood, The Danish Paediatrics Asthma Centre, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 2 Center for Applied Genomics and Division of Human Genetics, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; 3 Population Pharmacogenetics Group, Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom Correspondence and requests for reprints should be addressed to Hans Bisgaard, M.D., D.M.Sci, Copenhagen Studies on Asthma in Childhood, The Danish Paediatrics Asthma Centre, Faculty of Health Sciences, University of Copenhagen, Gentofte Hospital, Ledreborg Alle 34, DK-2900 Gentofte, Copenhagen, Denmark. E-mail: Bisgaard{at}copsac.dk
Rationale: An asthma predisposition locus on chromosome 17q12-q21 has recently been replicated in different ethnic groups. Objectives: To characterize the asthma and atopy phenotypes in early childhood that associate with the 17q12-21 locus. Methods: The single nucleotide polymorphism (SNP), rs7216389, was genotyped in 376 of 411 children from the Copenhagen Prospective Study on Asthma in Childhood (COPSAC) birth cohort born to mothers with asthma together with 305 mothers and 224 fathers. Nineteen additional SNPs in the region were genotyped in the children. Investigator-diagnosed clinical endpoints were based on diary cards and clinic visits every 6 months and at acute symptoms from birth. Lung function, bronchial responsiveness, and sensitization were tested longitudinally from early infancy. Measurements and Main Results: rs7216389 was significantly associated with the development of wheeze (hazard ratio 1.64 [1.05–2.59], P value = 0.03), asthma (hazard ratio, 1.88 [1.15–3.07], P = 0.01), and acute severe exacerbations (hazard ratio 2.66 [1.58–4.48], P value = 0.0002). The effect on wheeze and asthma was observed for early onset but not late onset of disease. The increased risk of exacerbations persisted from 1 to 6 years of age (incidence ratio 2.48 [1.42–4.32], P value = 0.001), and increased bronchial responsiveness was present in infancy and at 4 years of age, but not at 6 years. In contrast, rs7216389 conferred no risk of eczema, rhinitis, or allergic sensitization. Conclusions: Variation at the chromosome 17q12-q21 locus was associated with approximately twofold increased risk of recurrent wheeze, asthma, asthma exacerbations, and bronchial hyperresponsiveness from early infancy to school age but without conferring risk of eczema, rhinitis, or allergic sensitization. These longitudinal clinical data show this locus to be an important genetic determinant of nonatopic asthma in children.
Key Words: polymorphism • asthma • child • exacerbations • hyperresponsiveness • ORMDL3
Asthmatic symptoms represent the most common chronic illness in infants and preschool children (1). Hospitalization and other health care use are highest in preschool children, reflecting inadequate disease control (2, 3). Likewise, clinical trials often show incomplete control of exacerbations in young children compared with older children (4, 5). Together this suggests heterogeneity of the underlying disease with the very common asthmatic symptoms in early life probably reflecting different diseases with little to differentiate their clinical presentations (6). The discovery of new asthma genes opens the possibility of defining phenotypes from causal characteristics, which may improve prevention and treatment of this difficult to control disease entity. A recently reported genome-wide association (GWA) study of 317,000 single-nucleotide polymorphisms (SNPs) in a cohort of children with asthma from the U.K. and Germany resulted in the identification of a novel susceptibility locus on chromosome 17q12-q21 (7). Multiple-associated markers defined an interval that spanned 206 Kb and contained 19 genes. Differential expression of one gene, ORMDL3, was highly correlated with the asthma-associated SNPs, notably rs7216389, leading the authors to suggest ORMDL3 as the most likely disease candidate. The ORMDL genes encode transmembrane proteins anchored in the endoplasmic reticulum, but their physiological role is unknown (8). We recently replicated the association in North American white subjects (9) as have others in Scottish, French Canadian, African American, Puerto Rican, Mexican, and Japanese populations (10–13). The original observation suggested that the associated phenotype was particular to early-onset asthma, which was recently confirmed (14). The longitudinal impact on phenotypic expression in preschool-aged children has not previously been studied. Using this well-replicated asthma predisposition locus on chromosome 17q12-q21, we described the associated asthma and atopic phenotypes assessed longitudinally from birth to school age. We analyzed rs7216389 against the longitudinal clinical assessments of recurrent wheeze, asthma, acute severe exacerbations, eczema, and rhinitis, together with objective measurements of lung function, bronchial responsiveness, and sensitization from early infancy to school age in the high-risk birth-cohort "Copenhagen Prospective Study on Asthma in Childhood" (COPSAC) (15–18). None of the results of these studies has been previously reported.
The COPSAC birth cohort study is a prospective clinical study of a birth cohort of 411 infants born to mothers with a history of asthma. The newborns were enrolled at the age of 1 month, the recruitment of which was previously described in detail (15–18). The study was approved by the Ethics Committee for Copenhagen (KF 01-289/96) and The Danish Data Protection Agency (2008-41-1754) and informed consent was obtained from both parents. The families used doctors employed at the clinical research unit, and not the family practitioner, for diagnosis and treatment of any respiratory or skin-related symptoms. Participants were assessed at the COPSAC clinical research unit at six monthly intervals; additional visits were arranged immediately upon the onset of symptoms. At every visit, the infants were given a full physical examination, and history was obtained using structured questions and closed response categories focusing on the child's lung and skin symptoms, medication, healthcare use, lifestyle, and home environment.
Investigator-Diagnosed Clinical Endpoints Asthma was diagnosed according to the international guidelines, as previously detailed (17), and was based on recurrent wheeze as defined above. The character of symptoms, judged by the clinical research unit doctor, were considered to be typical of asthma with discrete exacerbations, but they also included symptoms between episodes, such as exercise-induced symptoms, prolonged nocturnal cough, persistent cough outside common cold, symptoms causing wakening at night (recently termed "multi-trigger wheeze" [19]) and in need of intermittent rescue with inhaled β2-agonist and responding to a 3-month course of inhaled corticosteroids but relapsing when stopping treatment. Episodic viral wheeze (19) is characterized as children who suffer from discrete wheezy episodes, feeling well between episodes, and treated intermittently with inhaled β2-agonist only. Temporal patterns of wheeze were defined as "early transient" if the child fulfilled the above criteria for recurrent wheeze in the first 3 years of life but not thereafter, "persistent" if the child also fulfilled the criteria from 5 to 6 years of age, and "late onset" if the child had a debut after 3 years of age. Acute severe exacerbations were defined from need of oral prednisolone or high-dose inhaled corticosteroid for wheezy symptoms prescribed at the discretion of the doctor at the clinical research unit or acute hospitalization at the local hospital for such symptoms. Rhinitis was diagnosed in children by 6 years of age by the clinical research unit doctor and was based on symptoms during the previous year of sneezing or a runny or blocked nose in periods when the child did not have a cold or flu (20).
Eczema.
Treatment Algorithms
Objective Measurements Bronchial responsiveness in infants was determined as the responsiveness to methacholine. The aerosol was administered with a dosimeter. After initial inhalation of saline, methacholine chloride was given in quadrupling dose steps from 0.04–16.67 µmol. The test procedure aimed at 20% fall in FEV0.5 or reaching the maximum dose. FEV0.5 was chosen as the endpoint for the baseline lung function and PD15(TcO2) as endpoint for bronchial responsiveness based on previous sensitivity analyses of known indices (23). Specific airway resistance (sRaw) was measured at 4 and 6 years by whole body plethysmography (24, 25). Bronchial responsiveness at the ages of 4 and 6 years was determined as the relative change in sRaw after hyperventilation of cold–dry air (26). Atopic sensitization was determined from specific IgE at the ages of 6 months, 1.5 years, 4 years, and 6 years by ImmunoCAP (27) (Phadia AB, Uppsala, Sweden) against the most common food and inhalant allergens (28). Values of 0.35 kU/L or greater were analyzed as the dichotomized index of any sensitization.
Genotyping
Multiple SNP genotyping.
Statistical Analyses For the transmission disequilibrium testing (TDT) parental discordance testing was performed as described (37) considering transmissions from heterozygous mothers versus heterozygous fathers to affected offspring separately. Analyses were done using R version 2.7.0 (www.r-project.org) and SAS version 9.1 (SAS Institute, Cary, NC).
The rs7216389 SNP was genotyped in 376 of 411 children who had good quality DNA available. Genotyping success rate was greater than 98%. The genotype distribution was: CC, 23%; CT, 48%; TT, 29%. The clinical follow-up rate of the COPSAC cohort was 95% at age 1 year; 90% at age 2 years; 85% at age 3 years; 79% at age 4 years; and 76% at age 5 and 6 years.
Investigator-Diagnosed Clinical Endpoints
Asthma.
Acute severe exacerbations. Fifty-seven of 376 children developed acute severe exacerbations on one or more occasions during follow-up. The Kaplan-Meier curves suggest a recessive genetic model with increased risk in subjects with the TT genotype (Figure EB). A log-rank test comparing CC and CT genotypes yielded a P value of 0.81. The overall HR in a recessive model for the T allele was significantly increased (2.66 [1.58–4.48]; P value = 0.0002). The Kaplan-Meier curves suggest a stable effect during the time of follow-up. Accordingly, the HR increased 2.5-fold during ages 0 to 3 years (2.46 [1.38–4.38]; P value = 0.002) and over threefold during ages 3 to 6 years (3.73 [1.14–12.23]; P value = 0.03). The population attributable cumulative 6-year risk in the recessive model was estimated to be 16.5 – 11.8 = 4.6%. The yearly incidence of having at least one wheezy exacerbation requiring high-dose steroid intervention or hospitalization per child in the study during the first 6 years of life is depicted in Figure 2 for the three genotypes. A recessive model could be assumed for age-adjusted incidence ratios (P value = 0.61). The incidence ratio for the TT versus CC or CT genotypes was 2.73 [1.49–5.00] (P value = 0.001).
Sensitization from 6 months to 6 years (37% of children) did not modify the effect on "asthma-related events" (P = 0.98). We also analyzed the association of the TT variant with three traditional subgroups of "wheezers" based on (1) temporal pattern of symptoms (31) showing a significant association to early transient but not late onset or persistent wheeze; (2) "atopic wheeze," i.e., wheeze with concomitant sensitization suggesting nonsignificant trends of association predominantly with the nonsensitized phenotype; and (3) the "episodic viral wheeze" (19) showing the odds of β2-agonist use was also significantly increased in children with TT genotype (OR = 1.62 [1.08–2.43], P value = 0.02) (see Table E1 in the online supplement).
Atopy.
Objective Endpoints.
Parental and Family-based Analyses In addition to genotyping the rs7216389 SNP in 376 COPSAC children we also genotyped it in 305 COPSAC mothers and 224 COPSAC fathers. Of those, 316 unrelated individuals had physician-diagnosed asthma during childhood. To determine if this phenotype also associates with the ORMDL3 locus, we performed a case-control analysis of the unrelated affected COPSAC individuals indicated, using the publically available genotypes from the 1958 birth cohort (n = 1500) (32) as controls. We performed Eigenstrat analysis on the sample set to control for population stratification. We observed a significant association of rs7216389 with asthma (minor allele frequency in cases 53% and 46% in controls; P value = 0.0002; OR = 1.3), thereby corroborating the original finding of Moffat and colleagues (7) to physician-diagnosed asthma in the Danish population. Because genotypes for the at-risk allele were available for the COPSAC family trios, we examined the parent-of-origin effect through transmission disequilibrium testing in the COPSAC children. We observed over-transmission of the at-risk T allele from the affected mothers to the affected offspring (maternal minor allele C transmitted:untransmitted 12.5:21.5; paternal minor allele C transmitted:untransmitted 14.5:14.5). Although preliminary, these results lend support to previous reports suggesting maternal origin of allelic effects in asthma and other atopic disorders (33).
Extended SNP Analyses at the ORMDL3 Locus
Key Results Here, we define for the first time, the asthma and atopy phenotype associated with the gene variants at the ORMDL3 locus on 17q12-q21 based on prospective longitudinal assessment of asthma symptoms and lung function from birth. In the COPSAC birth cohort, children homozygous for the T allele of rs7216389 were phenotypically characterized by early onset of asthma symptoms and increased risk of severe exacerbations as well as bronchial hyperresponsiveness assessed objectively from infancy to school age. No effect was observed on bronchial responsiveness or lung function at school age (6 years) or on eczema, allergic rhinitis, or allergic sensitization. This is the first example of a genetically defined nonatopic asthma phenotype of the early childhood. Asthma symptoms and asthma exacerbations in early life represent a severe disease burden with major impact on quality of life for patients and socioeconomic costs for the health care system. The TT genotype of the rs7216389 variant at the ORMDL3 locus was strongly associated with recurrent wheeze, asthma, severe asthma exacerbations, and hospitalization from infancy. Furthermore, the population impact of this genetic polymorphism was substantial due to the high allele frequency of the disease variant. The population attributable cumulative 6-year risk suggests that elimination of the risk associated with the T allele in a similar population of children born to mothers with a history of asthma should reduce the proportion of children with asthma and severe exacerbations before the age of 6 years by 16 and 28%, respectively. The effect of the at-risk allele of rs7216389 on the physician-diagnosed asthma phenotype, as originally reported by Moffat and colleagues (7), was also observed in the Danish population in a case-control analysis of mothers with asthma in the COPSAC birth cohort compared with publically available genotypes from the 1958 birth cohort (32). The strength of the association here is in keeping with the original report and our recent replication (9). Finally, several tagging SNPs extracted from a genome-wide genotyping analysis for the 17q12-q21 locus were also strongly associated with the same phenotypes as the rs7216389 variant, thereby further substantiating the association signals in the COPSAC children.
Strength and Limitations A well-defined phenotype is essential in genetic association studies. This is particularly difficult in the clinical evaluation of the early childhood wheeze where interobserver variation is a significant problem due to inaccurate use of terms among clinicians and caregivers (34–37). The major strength of the COPSAC study is the meticulous prospective clinical monitoring, diagnosing, and treatment of lung and skin symptoms based on standard operating procedures by the investigators from this single clinical research unit through the first 6 years of life of the cohort. The cohort was seen regularly at 6-month intervals as well as for acute lung and skin manifestations by the doctor in the COPSAC clinic, who controlled diagnosis and treatment according to predefined algorithms, (i.e., diagnoses and treatments were not made by doctors outside our research unit). Additionally, the longitudinal objective assessments of lung function and bronchial responsiveness from birth through preschool age assure robust objective endpoints. Together this prospective clinical monitoring in a single center is the key difference to other cohorts often based on questionnaires and parents history of diagnoses made by doctors in the community. Asthma was diagnosed prospectively at the clinical research unit according to a rigid algorithm based on predefined recurrence of diary-recorded wheezy episodes, symptoms typical of asthma, need of short-acting bronchodilator treatments, response to inhaled corticosteroids, and relapse after stopping treatment (17). The accuracy of such pragmatic diagnosis was strengthened by the history being reported by mothers all experienced with asthma and by consistency with the independent intermediary endpoints recurrent wheeze and acute severe exacerbations as well as the repeated objective assessments of bronchial hyperresponsiveness. The power of the statistics was improved from the longitudinal data set with the time of onset clearly distinguishing these populations. Complex human diseases have variable ages of onset. Because the age of onset is likely to be genetically mediated, the subject's age of onset carries more information about the etiology of the disease than the case-control status. Cross-sectional analyses have less statistical power. The external validity of this study is limited by the high-risk nature of the cohort and would benefit from replication in population-based studies. Atopic sensitization did not modify the association between rs7216389 and asthma-related events suggesting that the increased frequency of sensitization in this cohort did not modify the effect of the gene variant. Although the parent of origin transmission disequilibrium testing in COPSAC demonstrated overtransmission of the risk T allele from the affected mothers to the affected offspring, our study is too small to accurately estimate the risk ratio of such overtransmission from the affected parent. Data from both the initial discovery study and a follow-up association study in African Americans, Puerto Ricans, and Mexicans is suggestive of genetic complexity at the 17q12-q21 interval with the possibility of multiple independent effects in Northern Europeans and varying patterns of association in the non-European populations. In line with the discovery study (7), we showed significant linkage disequilibrium between several SNPs in the region and our results confirm rs7216389 as a relevant tagging SNP in populations of Northern European ancestry. We used this surrogate to tag at least one of the major asthma predisposition variants at this locus.
Interpretation Different phenotypes of young children with asthma-like symptoms have been proposed based on the temporal patterns of symptoms and their relation to markers of atopy of which the TT genotype of the rs7216389 variant resembles mostly "transient wheezer" (31) and "the nonatopic wheezing phenotype" (40). However, phenotyping based on symptom course and relation to atopy have had little influence on the treatment of preschool wheeze and the understanding of the underlying pathogenesis, whereas phenotypes defined from genotypes are likely to improve targeted treatment and prevention because of the causal relation. Therefore, the recognition of this common gene variant as a major risk factor for nonatopic asthma and severe exacerbations in young children of Northern European descent has the potential of being an important step for the urgently needed improved treatment and prevention of this disease as well as research into environmental risk factors. Bronchial responsiveness was increased in newborns and maintained during preschool age in children homozygous for the T allele of the rs7216389 polymorphism. This lends biological plausibility to the findings of increased exacerbations rate and suggests that increased bronchial responsiveness is a hallmark and maybe a causal intermediate step between this genetic variation and symptoms of the associated asthma phenotype. In contrast, the gene variant was not associated with bronchial responsiveness or lung function in children at 6 years of age. This is in line with a recent study suggesting that reduced lung function at school age is associated with the atopic and not the nonatopic asthma phenotype (41). The underlying mechanism of the association between genetic variation on chromosome 17q12-q21 and asthma is not yet understood. Multiple variants within the region have been shown by us and others to contribute independent effects, indicating the potential for several functional SNPs in the region. Among the potentially functional variants within the interval, rs7216389, which lies within an intron of the GSDML gene, was shown to be associated with the transcript levels of the neighboring ORMDL3 gene. Although the functional effects of the disease-associated variant of rs7216389 on ORMDL3 expression renders it a strong candidate for the disease-causal variation, one needs to keep in mind that the association between rs7216389 and ORMDL3 expression may be coincidental to asthma predisposition and one or more of the other associated SNPs in the interval may underlie the reported association with the disease trait. Resequencing of the region will be needed to pinpoint the actual functional variant(s). The discovery cohort of adults recalling "asthma ever" reported an OR of 1.21 [1.04–1.40]. Recall of "asthma attacks" before the age of 8 years was associated with rs3894194 (OR 1.68 [1.25–2.26]) (7). The increased risk of early childhood asthma and exacerbations was confirmed in recent studies (10–12, 14). Our data are consistent with these observations, and, by longitudinal assessment from birth, we extended the observations by showing a strong association with severe symptoms from the first year of life. The recessive model for the T allele of the rs7216389 SNP was appropriate for all objective and clinical outcomes. A current case-control study also reports a T allele recessive model for asthma exacerbations but finds a T allele dominant model for asthma in a cross-sectional study of 3- to 22-year-old individuals (10). It is plausible that differences in the genetic mechanism may vary between disease outcomes and over time, and that heterozygous CT individuals in our cohort may develop asthma more frequently than those homozygous for the C allele.
Conclusions
The authors thank the children and parents participating in the COPSAC cohorts as well as the COPSAC study teams.
The COPSAC is supported by private and public research funds. Grants greater than 100,000 Euro were received from the Lundbeck Foundation, the Pharmacy Foundation of 1991, Augustinus Foundation, the Danish Medical Research Council, and the Danish Pediatric Asthma Centre. The Children's Hospital of Philadephia funded genome-wide genotyping of the COPSAC cohort through an Institutional Development Fund grant to the Center for Applied Genomics (H.H.). 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.200809-1436OC on November 21, 2008 Conflict of Interest Statement: H.B. has been a consultant and advisor to NeoLab, Nycomed, and Merck and paid lecturer for AstraZeneca and Merck; he holds sponsored grants from NeoLab and Medimmune. K.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.M.A.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.K.-M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.B.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.N.A.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form September 11, 2008; accepted in final form November 21, 2008
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