Abstract
Rationale Substantial variability in response to asthma treatment with inhaled corticosteroids (ICS) has been described among individuals and populations, suggesting the contribution of genetic factors. Nonetheless, only a few genes have been identified to date. We aimed to identify genetic variants associated with asthma exacerbations despite ICS use in European children and young adults and to validate the findings in non-Europeans. Moreover, we explored whether a gene-set enrichment analysis could suggest potential novel asthma therapies.
Methods A genome-wide association study (GWAS) of asthma exacerbations was tested in 2681 children of European descent treated with ICS from eight studies. Suggestive association signals were followed up for replication in 538 European asthma patients. Further evaluation was performed in 1773 non-Europeans. Variants revealed by published GWAS were assessed for replication. Additionally, gene-set enrichment analysis focused on drugs was performed.
Results 10 independent variants were associated with asthma exacerbations despite ICS treatment in the discovery phase (p≤5×10−6). Of those, one variant at the CACNA2D3-WNT5A locus was nominally replicated in Europeans (rs67026078; p=0.010), but this was not validated in non-European populations. Five other genes associated with ICS response in previous studies were replicated. Additionally, an enrichment of associations in genes regulated by trichostatin A treatment was found.
Conclusions The intergenic region of CACNA2D3 and WNT5A was revealed as a novel locus for asthma exacerbations despite ICS treatment in European populations. Genes associated were related to trichostatin A, suggesting that this drug could regulate the molecular mechanisms involved in treatment response.
Abstract
A genome-wide association study of asthma exacerbations despite inhaled corticosteroid treatment in childhood asthma revealed a novel association at the CACNA2D3-WNT5A locus and suggested trichostatin A as a potential asthma therapy https://bit.ly/3nxWLPD
Introduction
Asthma is the most common chronic condition in children and young adults [1]. Inhaled corticosteroids (ICS) are the first-line treatment recommended by current international guidelines to control and prevent asthma symptoms [1]. Although ICS are the most effective medication for improving symptoms and preventing severe exacerbations [2], high inter-individual variability in ICS response has been described [3]. Studies have shown that 30–40% of the asthmatic children treated with ICS do not show an improvement of their symptoms and that 10–15% of them may even experience worsening of asthma exacerbations despite the regular use of this medication [3]. Moreover, marked variation in ICS response has been described among populations [4].
The contribution of genetic factors in asthma-related traits has been widely suggested [5]. Specifically, the variation in ICS response has been suggested to be the result of the interaction of several factors such as the specific asthma endotype, comorbidities, ancestry, the environment and the individual's genetic composition [6]. Approximately 40–60% of the total variation in ICS response may be explained by genetic factors [7]. Pharmacogenetic studies of ICS response have focused mostly on a few genes with known biological implications in the mechanisms of action of ICS [5]. More recently, genome-wide association studies (GWAS) have explored the role of genetic variation in ICS response [8–10]. Overall, these GWAS have identified 13 genes associated with different definitions of ICS response, most of which were not previously associated with asthma-related phenotypes, except for PDE10A [11]. However, it is expected that more genes are involved in the response to this asthma treatment. Moreover, the genetic architecture of clinical markers of disease severity, such as asthma exacerbations or lung function measurements, is not completely disentangled [12, 13]. The studies performed to date have been limited by the relatively small number of study participants. Therefore, there is a need for studies including a large number of individuals to increase the power to detect significant associations with asthma severity and ICS response [5]. Increasing the knowledge about the genetic markers involved in asthma progression and therapeutic response would be of special importance in clinical practice since current international guidelines for the management of asthma propose pharmacological stepwise approaches based on the occurrence and persistence of clinical outcomes as indicators of disease severity [1].
In the present study, we aimed to replicate suggested associations in a candidate gene approach and to identify novel genetic variants involved in the occurrence of asthma exacerbations despite ICS treatment by performing a large GWAS in Europeans and to examine whether this genetic variation is shared with other populations. In addition, we explored whether a gene-set enrichment analysis of the GWAS results could suggest treatments that could be potential therapeutic alternatives in patients who do not respond to ICS therapy.
Methods
Ethics statement
All studies included were approved by their local institutional review boards and written informed consent was provided by participants or their parents/caregivers. All methods were carried out following guidelines and regulations for human subject research under the principles of the Declaration of Helsinki.
Study populations
A total of 14 independent studies participating in the Pharmacogenomics in Childhood of Asthma (PiCA) consortium [14] were included in this study. Eight available studies in populations of European descent at the time of data collection were included in the discovery phase, whereas replication of association results was evaluated in three additional independent European studies. Further validation was performed in three non-European studies from Hispanic/Latino, African American and Asian populations.
Discovery phase
Asthma patients from eight independent European studies were analysed in the discovery phase: the Pharmacogenetics of Asthma Medication in Children: Medication with Anti-inflammatory Effects (PACMAN); the Paediatric Asthma Gene–Environment Study (PAGES); BREATHE; the Genetics of the Scottish Health Research Register (GoSHARE); the Pharmacogenetics of Adrenal Suppression with Inhaled Steroids study (PASS); SLOVENIA; the follow-up stage of the Multicenter Asthma Genetics in Childhood Study (followMAGICS); and Effectiveness and Safety of Treatment with Asthma Therapy in Children (ESTATe). All these studies included children and young adults aged 2–25 years recruited in five different European countries. Among the participants, only individuals with reported use of ICS, information about asthma exacerbations and genome-wide genotyping data were included. ICS use was based on declared use of any type of ICS and/or combination with long-acting β2-agonists at least once in the previous 12 months based on self-reports, pharmacy or medical records [15]. A period of the past 6 months was considered for those studies without data available related to the previous year. A detailed description of each study is provided in the supplementary material.
The presence or absence of at least one asthma exacerbation episode during the 6 or 12 months preceding the study enrolment was assessed. Severe asthma exacerbations were defined by a need for emergency care, hospitalisations or administration of systemic corticosteroids because of asthma for PACMAN, GoSHARE, PASS, SLOVENIA and ESTATe (table 1) [16]. Definitions of moderate asthma exacerbations were used in BREATHE-PAGES, BREATHE and followMAGICS (table 1), since no information was available for any of the previous variables [16]. Therefore, data related to unscheduled general practitioner or respiratory system specialist visits and school absence were also considered in the definition of asthma exacerbations for BREATHE-PAGES, BREATHE and followMAGICS (table 1), as described elsewhere [15].
Clinical and demographic characteristics of the European populations included in the discovery phase
Replication phase
Validation of the results found in the discovery phase was carried out in three independent European studies: the Avon Longitudinal Study of Parents and Children (ALSPAC); the Childhood Asthma Management Program (CAMP) and, the Children Allergy Milieu Stockholm an Epidemiological Study (BAMSE). Definitions of ICS use and asthma exacerbations were based on retrospective information about the 12 months prior to study enrolment adopting the same criteria applied in the discovery phase, except for prospective data from CAMP. Further details about these studies are described in the supplementary material.
Assessment of ICS associations in non-European populations
Association signals with evidence of replication (p≤0.05) among Europeans were evaluated in Latino/Hispanic subjects from the Genes-Environment and Admixture in Latino Americans (GALA II) study, African American subjects included from the Study of African Americans, Asthma, Genes, and Environments (SAGE), and Asian subjects from the Singapore Cross-Sectional Genetic Epidemiology Study (SCSGES). Information about the presence or absence of asthma exacerbations despite ICS use in the 12 months prior to study enrolment was considered. The details on these studies are described in the supplementary material.
Genotyping, genetic ancestry estimation and imputation
Samples from the studies included in the discovery phase were genotyped using different platforms for previous studies (table 1) [15], except for PAGES, GoSHARE and part of the samples from BREATHE. These studies were genotyped using the Axiom Precision Medicine Research Array (Affymetrix, Santa Clara, CA, USA) by Centro Nacional de Genotipado (CeGen; www.cegen.org). The same quality control procedures described in Hernandez-Pacheco et al. [15] were applied to all the studies. Further details are available in the supplementary material.
Details about the genotyping of the replication samples are provided in the supplementary material and summarised in supplementary table S1. Similarly, the genotyping methods used for the non-European studies are described in supplementary table S2.
Assessment of the genetic ancestry was carried out through principal component (PC) analyses or by model-based assessments of the proportions of genetic ancestry (GALA II and SAGE) [15]. For SCSGES, estimation of ancestry was not performed, since genome-wide genotyping was not available. The second release of the Haplotype Reference Consortium (r1.1 2016) was used as reference panel for imputation [17], except for CAMP and ALSPAC, where phase three of the 1000 Genomes Project was used [18].
Association analysis in the discovery phase
GWAS analyses were carried out separately for each study, except for PAGES and a subset of individuals from BREATHE that were genotyped together with PAGES. These two studies were analysed together since the similarities of the study design, type of biological samples, demographic and clinical characteristics, and genotyping platform used, and are denoted as BREATHE-PAGES. Association between genetic variants and the binary variable of asthma exacerbations was tested employing the binary Wald logistic regression model implemented in EPACTS 3.2.6 [19]. Regression models included as covariates age, sex and the PCs needed to control for population stratification within each study.
Results for single nucleotide polymorphisms (SNPs) with a minor allele frequency (MAF) ≥1% and imputation quality (r2) ≥0.3 obtained for each study included in the discovery phase were meta-analysed. Fixed-effects or random-effects models were applied using METASOFT [20], depending on the significance of the Cochran Q-test evidencing heterogeneity among the studies analysed. Association with asthma exacerbations despite the use of ICS treatment was considered at suggestive significance level (p≤5×10−6), which was arbitrarily set based on the criteria commonly adopted in GWAS studies [15].
Independent association signals were detected from these results through conditional and joint multiple-SNP analyses, as implemented in Genome-wide Complex Trait Analysis 1.92.0 [21]. Stepwise model selection was carried out to select independently associated SNPs within each genomic region with a suggestive association signal through a linkage disequilibrium correlation matrix obtained with the data from PACMAN, the largest study included in the discovery phase. Independent SNPs associated (p≤5×10−6) with asthma exacerbations were followed-up for replication.
Association analysis in the replication phase
Association analyses were performed in three different PiCA studies of European descent. The definition of asthma exacerbations used for each replication population is described in supplementary table S1. Association testing in BAMSE was performed following the same methodology as in the discovery phase. Logistic regressions were carried out in CAMP and ALSPAC using PLINK 1.9 [22] and SNPTEST 2.5.2 [23], respectively. Association results obtained from the European replication studies for variants associated with asthma exacerbations despite ICS use at nominal level (p≤0.05), and with the same direction of the effects as in the discovery phase were meta-analysed following the same methodology as described earlier.
Association analysis in non-European populations
The association of the variant with evidence of replication was further assessed in GALA II and SAGE using the same statistical methodology applied for the studies included in the discovery phase. In SCSGES (supplementary table S2), association with asthma exacerbations was evaluated using logistic regressions adjusted by age and sex using PLINK 1.9 [22].
Evidence of validation was considered if the variant assessed showed a p-value ≤0.05 and the same direction of the effect as the one found in European populations.
Association analysis accounting for ICS dosage and asthma severity
Several sensitivity analyses were performed to ascertain whether the effect of the associations found in different populations was driven by potential confounders of the response to asthma medication or disease severity. Specifically, association analyses with asthma exacerbations were performed for the variant with evidence of replication. First, logistic regressions were carried out evaluating the association with the presence/absence of asthma exacerbations accounting for the daily ICS dosage in PACMAN, the largest study with available information for this variable, as described in the supplementary material. Additionally, association analyses were carried out accounting for asthma severity based on the classification into treatment steps based on a modification of the guidelines established by the British Thoracic Society (BTS) and the Scottish Intercollegiate Guidelines Network (SIGN) [24]. Only those individuals with available information about the use of the medications included in the classification into treatment steps were selected and they were classified as described in the supplementary material.
In silico functional evaluation of variants associated with asthma exacerbations despite ICS use
Functional evaluation of the variant with evidence of replication was carried out using publicly available databases. Evaluation of functional evidence described in the Encyclopedia of DNA Elements (ENCODE) was used to assess the role as expression quantitative trait loci (eQTL), DNase hypersensitivity sites and histone marks using HaploReg v4.1 [25], and the Portal for the Genotype-Tissue Expression was also queried [26]. Previous significant evidence as protein quantitative trait loci (pQTL) or methylation quantitative trait loci (meQTL) was also explored using publicly available information by means of the PhenoScanner v2 tool [27, 28].
Validation of previously reported ICS genes in European populations
Previous studies identified a total of 26 SNPs located near or within 15 genes associated with ICS response in different populations (supplementary table S3). These variants were analysed in the present dataset using the meta-analysis results of the discovery phase of the current GWAS.
Validation of previous associations was performed at the SNP level, searching for consistent association at the nominal level (p≤0.05). Additionally, replication was also assessed as genomic regions, analysing variants located within 100 kb upstream and downstream from the gene limits. A Bonferroni-corrected significance threshold was estimated for each genomic region as α=0.05 per number of independent variants analysed, using the same methodology as described elsewhere [15].
Enrichment analysis of drug targets
A gene-set enrichment analysis focused on drugs was performed using the summary association results from the discovery phase of this GWAS. An overlap between the genes associated with asthma exacerbations in the discovery phase and gene sets with previous evidence of expression inhibition or induction after exposure to drugs or small molecules was inspected. For that, variants were first assigned to the nearest gene using the UCSC Table Browser tool [29]. Not only were SNPs associated (p≤5×10−6) with asthma exacerbations despite ICS treatment in the discovery phase included, but those significant at p≤1×10−4 were also analysed to increase the statistical power to detect genes previously identified to show drug-induced changes in expression levels. This threshold was arbitrarily set as it is commonly carried out in gene-set enrichment approaches [30, 31]. For this analysis, the information available at the Drug Signatures Database and DrugMatrix was used utilising the Enrichr tool [32]. Evidence of significant enrichment at drugs was considered for those genes with significant drug-related expression changes after accounting for the multiple comparisons tested (false discovery rate (FDR) ≤0.05).
Results
Characteristics of the study populations
2681 children and young adults with asthma from eight studies were analysed in the discovery phase (table 1), whereas 538 patients from different populations were included in the replication stage of this GWAS in Europeans (supplementary table S1). Individuals from the studies analysed in the discovery phase showed a similar mean age, except for followMAGICS, which included individuals with older ages (17.2±3.0 years) (table 1). Although different definitions of asthma exacerbations were used, similar proportions of exacerbations were found across European populations included in the discovery phase, except for PACMAN and GoSHARE, which showed the lowest asthma exacerbations rates (11.0% and 13.8%, respectively) (table 1). Among the non-European samples, Latino/Hispanic subjects from GALA II had the highest proportion of asthma exacerbations occurrence despite the treatment with ICS (66.4%) (supplementary table S2).
Association results in European populations
Association results for a total of 8.1 million common SNPs (MAF ≥1%) with r2≥0.3 and shared among the eight European populations included in the discovery phase were meta-analysed. No major evidence of genomic inflation due to population stratification was found when each study was individually analysed (supplementary figure S1a–h), nor after combining them in a meta-analysis (λGC=1.04) (supplementary figure S1i). Although no associations were detected at the genome-wide significance level (p≤5×10−8), a total of 19 variants near or within 10 loci showed p≤5×10−6 in European children and young adults (supplementary table S4; figure 1). Among those polymorphisms, one independent variant per locus was found after performing pairwise regressions conditioned on the most significant variant for each locus with more than one association signal. Thus, a total of 10 independent signals were detected (table 2), which were followed-up for replication.
Manhattan plot of association results of asthma exacerbations in inhaled corticosteroid users included in the discovery phase. Association results are represented as −log10 p-value on the y-axis along the chromosomes (x-axis). The horizontal black line shows the suggestive significance threshold for replication (p≤5×10−6).
Summary of the conditional regression models for each locus suggestively associated with asthma exacerbations in patients treated with inhaled corticosteroids (ICS) in the discovery phase
Of the 10 variants associated with asthma exacerbations despite ICS treatment in the discovery phase (p≤5×10−6), only the SNP rs67026078, located within the intergenic region of CACNA2D3 and WNT5A (figure 2), showed nominal replication after meta-analysing the European studies included in the replication (OR for C allele 1.83, 95% CI 1.16–2.90; p=0.010) (table 3). The association had a consistent effect as in the discovery phase (OR for C allele 1.50, 95% CI 0.93–2.43; p=4.22×10−6) (table 3). Suggestive genome-wide association was found for this SNP after performing a meta-analysis across the European studies analysed in both phases (OR for C allele 1.58, 95% CI 1.11–2.26; p=4.34×10−7) (figure 3). Nonetheless, the association effect of this variant was mostly driven by the studies with information about the occurrence of asthma exacerbations available for a 12-month period. This could be explained by the fact that a wider timeframe makes exacerbation events likely to occur, but also by the larger sample size analysed compared to the studies with information based on the previous 6 months (n=1557 versus n=1124).
Regional plot of association results for the CACNA2D3-WNT5A locus for the European populations included in the discovery phase. Logarithmic transformation of the association results (−log10 p-value) is represented in the y-axis by chromosome position (x-axis) for each single nucleotide polymorphism (SNP) as a dot. The SNP rs67026078 with evidence of replication in the European populations included in the replication phase is represented by a diamond. The remaining variants are grey colour-coded based on pairwise r2 values with that SNP for European populations from the 1000 Genomes Project.
Association results for the independent suggestive associations followed-up for replication in populations of European descent
Forest plot of association effect of rs67026078 across European studies included in the genome-wide association study of asthma exacerbations despite inhaled corticosteroid treatment. Association effects are shown in terms of odds ratio for the effect allele for each study and after meta-analysing the results from both phases by black boxes and a diamond, respectively. Effect of association results is not given for BREATHE, since rs67026078 did not pass quality control checks. PACMAN: Pharmacogenetics of Asthma Medication in Children: Medication with Anti-inflammatory Effects; PAGES: Paediatric Asthma Gene–Environment Study; GoSHARE: Genetics of the Scottish Health Research Register; PASS: Pharmacogenetics of Adrenal Suppression with Inhaled Steroids study; followMAGICS: the follow-up stage of the Multicenter Asthma Genetics in Childhood Study; ESTATe: Effectiveness and Safety of Treatment with Asthma Therapy in Children; ALSPAC: Avon Longitudinal Study of Parents and Children; CAMP: Childhood Asthma Management Program; BAMSE: Children Allergy Milieu Stockholm an Epidemiological Study.
Assessment of ICS associations in non-European populations
The SNP rs67026078 with evidence of replication in independent European populations was not associated with asthma exacerbations in patients treated with ICS from Hispanic/Latino nor African American populations (supplementary table S5). In Asian subjects, this variant was not consistently associated with asthma exacerbations in SCSGES (supplementary table S5). Differences in the effect allele frequency of this variant were found among the populations evaluated, being higher in the studies of European ancestry included in the discovery (6.1–9.3%) and replication (5.7–9.4%) phases, compared to the non-European populations. Specifically, this variant had a frequency of 4.7%, 4.9% and 1.4% in Hispanic/Latino, African American and Asian subjects, respectively.
Association analysis accounting for ICS dosage and asthma severity
Sensitivity analyses of asthma exacerbations despite ICS use including daily medication dosages as a covariate in 521 asthma patients of European descent from the PACMAN study revealed that the association effect of rs67026078 adjusted by the ICS did not account for the association with the occurrence of asthma exacerbations (OR for C allele 1.24, 95% CI 1.14–1.34; p=2.30×10−7). These results were equivalent in terms of significance to those obtained applying the original association model for the same individuals with complete data, but the effect sizes were smaller (OR for C allele 4.30, 95% CI 2.33–7.92; p=2.98×10−6 in the model not adjusted by ICS dose). Similar results were found adjusting by a categorical variable related to ICS dose based on age groups (OR for C allele 1.23, 95% CI 1.14–1.34; p=2.02×10−7) (supplementary table S6).
Association analyses adjusted by asthma severity based on treatment steps classification were performed in 2282 asthma patients from the discovery phase with available data related to the medication use (table 1). The SNP rs67026078 was suggestively associated with asthma exacerbations after accounting for disease severity (OR for C allele 1.43, 95% CI 0.88–2.33; p=1.05×10−5). These results are equivalent to those obtained applying the original association models to the individuals with available classification into treatment steps (OR for C allele 1.45, 95% CI 0.91–2.33; p=1.03×10−5).
Functional evaluation of the variant associated with asthma exacerbations despite ICS use
According to the ENCODE project, the SNP with evidence of replication among Europeans, rs67026078, is located within a histone H3 lysine 4 mono-methylation (H3K4me1) mark in several tissues, including fetal lung fibroblasts and other fetal pulmonary cells. Its suggestive role in regulating gene expression is also shown by the fact that this is a DNAse hypersensitivity site in lung fibroblast primary cells [33]. However, no evidence of significant eQTL was found for this SNP. Nonetheless, previously the SNP rs67026078 had been significantly identified (p≤0.01) as pQTL and meQTL. Specifically, B.B. Sun and co-workers found this variant to be associated with protein expression levels for 16 different proteins in plasma [27, 28, 34] (supplementary table S7). Some of these have been related to molecular and cellular processes related to asthma pathophysiology (ADAMTS5) and involved directly or indirectly in the Wnt pathway (PSMA2, ADAMTS5, ATAD2, CHST3, TEAD3) [35]. Moreover, rs67026078 was found to regulate the methylation patterns of a CpG site (cg16278514) at the intergenic region of CACNA2D3 and WNT5A in whole blood by M.J. Bonder and co-workers [27, 28, 36]. Interestingly, both CACNA2D3 and WNT5A are expressed in pulmonary tissues [26].
Validation of genes previously associated with ICS response
Among the 26 SNPs associated in previous GWAS of ICS response, one variant intergenic to UMAD1 and GLCCI1 (rs37972) showed evidence of replication in European populations included in the PiCA consortium (OR for C allele 1.20, 95% CI 1.05–1.37; p=6.58×10−3) (supplementary table S8). Considering the genomic regions where these genes reside, 33 096 variants located within 100 kb upstream and downstream from the 15 genes of ICS response previously described were evaluated. Accounting for the number of independent association signals within each genomic region, evidence of replication was found for 40 SNPs near five genomic regions: PDE10A-T (SNP with min p-value: rs57042153, OR for T allele 1.43, 95% CI 1.20–1.70; p=5.97×10−5), UMAD1-GLCCI1 (rs13235500, OR for G allele 0.71, 95% CI 0.60–0.85; p=2.44×10−4), SHB-ALDH1B1 (SNP with min p-value: rs341488, OR for A allele 2.24, 95% CI 1.48–3.40; p=1.44×10−4), ZNF432-ZNF841 (SNP with min p-value: rs67834224, OR for A allele 0.65, 95% CI 0.52–0.82; p=2.86×10−4), ELMO2-ZNF334 (SNP with min p-value: rs11087003, OR for C allele 0.77, 95% CI 0.66–0.89; p=5.84×10−4) (supplementary table S9). However, none of these associations were significant after correction for the total number of SNPs tested across all genomic regions (1799 independent SNPs: Bonferroni-like correction significance threshold of p≤2.78×10−5).
Enrichment analysis in European asthmatic children and young adults treated with ICS
Enrichment analysis of associations from the GWAS results focused on drugs was carried out, including 782 SNPs associated with asthma exacerbations despite ICS treatment (p≤1×10−4) in the discovery phase. A total of 49 different drugs and small molecules that had been found to regulate expression levels of the genes associated with asthma exacerbations in the GWAS were revealed (supplementary table S10). Of those, trichostatin A (TSA) remained statistically significant after adjusting for multiple comparisons (FDR=0.035) (supplementary table S10). Specifically, 30 of the 83 genes associated at p≤1×10−4 in our GWAS had been previously proposed as targets of TSA, since changes in expression levels were found to be triggered by the exposure to this drug (supplementary table S11). These genes included several loci previously associated with asthma-related traits and allergic diseases (e.g. RERE, NEGR1, ROBO2, LAMA2, SLC11A2, JMJD1C) or involved in drug metabolism (e.g. AOX1) (supplementary table S12) [35, 37].
Discussion
To our knowledge, this study describes the results of the largest GWAS of asthma exacerbations in children and young adults treated with ICS to date. After combining eight different studies of European ancestry, 10 independent variants were found to be suggestively associated with asthma exacerbations despite ICS treatment. One SNP within the intergenic region of CACNA2D3 and WNT5A showed evidence of replication at nominal level in three independent European populations. However, this was not validated in Latino/Hispanic, African American or Asian subjects, which could be due to ancestry-specific effects. Additionally, we found evidence of replication for five different genes associated with ICS response by previous GWAS studies at SNP or genomic-region level. Furthermore, an enrichment analysis of association signals with asthma exacerbations revealed TSA as a potential regulator of the molecular mechanisms involved in asthma pathogenesis.
CACNA2D3 encodes a member of the α-2/δ subunit family, which are voltage-dependent calcium channels consisting of a complex of α-1, α-2/δ, β and γ subunits. Specifically, CACNA2D3 modulates the calcium current density through the regulation of the influx of calcium ions into the cell upon membrane polarisation [38]. CACNA2D3 has important functions given the fact that calcium is a secondary messenger involved in multiple cellular processes such as cell proliferation, apoptosis, adhesion and migration [39]. This gene could have a role in respiratory diseases, since variants located near to CACNA2D3 have been recently associated with different lung function measurements, which are important predictors of asthma severity and progression [40, 41]. Specifically, these associations include forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC) and the FEV1/FVC ratio in COPD patients from the large cohort of European descent UK Biobank [42, 43], and the change in lung function after administration of bronchodilators in smokers [44]. It is well known that pulmonary function is an important predictor of asthma severity and progression [40, 41]. Additionally, an intronic CACNA2D3 variant (rs1820616) has been associated with the fractional concentration of nitric oxide in exhaled air [45], which is a good indicator of inflammatory patterns in the airways and a powerful approach to support asthma diagnosis in children [46] and to monitor the adherence and response to medications [47]. These findings suggest that CACNA2D3 could be involved in asthma progression, including the risk of asthma exacerbations, even in patients under ICS therapy.
WNT5A encodes for the WNT family member 5A, a lipid-modified glycoprotein that activates diverse signalling pathways [48]. This protein has been evidenced to play a crucial role in development during embryogenesis, oncogenesis, and regulation of inflammatory processes in infectious disorders [49]. Moreover, other genes encoding for ligands involved in the WNT signalling pathway are associated with impaired lung function in asthmatic children [50]. This suggests that WNT5A could be also involved in the pulmonary capacity in asthma. Interestingly, genes associated with asthma susceptibility have been linked to WNT signalling through a gene-set enrichment analysis [30]. Specifically, this biological process seems to play regulatory and suppressive roles through the modulation of inflammation and structural changes in airways. WNT ligands have been proposed to act on the major players implicated on inflammatory processes such as dendritic and T-helper type 2 (Th2) cells and macrophages [51]. Indeed, WNT molecules regulate the homeostasis of these cells, avoiding dysregulated immune responses, which could trigger several diseases, including allergic asthma [51].
Specifically, expression of WNT5A has been positively associated with Th2-mediated airway inflammation in asthmatic patients [52]. Additionally, eosinophils derived from asthma patients have been found to enhance expression levels of this gene in airway smooth muscle (ASM) cells, triggering cell proliferation, inflammatory processes and airway remodelling [53]. It is well known that eosinophilia at blood and tissue levels is one of the most important phenotypes in asthma patients [54], triggered by high levels of chemokines and cytokines. Specifically, eosinophils migrate from lymph nodes to the airway in asthma, where they adhere to the ASM, releasing transforming growth factor (TGF)- β1 molecules [55]. Increased levels of TGF-β1 have been related to the overexpression of WNT5A in ASM cells at gene and protein levels compared to healthy individuals. Therefore, production of extracellular matrix proteins is induced, increasing ASM mass and contractility and hence airway remodelling by means of hypertrophy and hyperplasia [53]. These findings suggest the important role of the WNT5A and the WNT signalling pathway in asthma pathogenesis, making it a promising therapeutic target in asthma [56], throughout inhibition of WNT ligands biogenesis, secretion and blocking their ligand–receptor interactions through small pharmacological molecules [49]. Nonetheless, further research is needed to explore the potential side-effects of drugs targeting this pathway, since tumorigenesis-related functions have been also widely attributed to WNT molecules [57].
The C allele of the SNP rs67026078, which is located 54.1 kb from the 3′ untranslated region of CACNA2D3, was found to be associated with an increased risk of asthma exacerbations despite the ICS treatment across the European studies analysed in the discovery and replication phases. Sensitivity analyses accounting for baseline asthma severity suggested that the effect of this association is related to the response to asthma medications or to the biological drivers of asthma exacerbations. Nonetheless, this was not shown to be significantly associated with asthma exacerbations in patients treated with ICS from Hispanic/Latino, African American or Asian populations. This result could be explained by ancestry-driven effects evidenced by the lower frequency of the effect allele of this variant in non-European populations. This polymorphism had not been previously associated with asthma treatment response, although functional evidence suggests that this variant could be actively involved in the regulation of gene expression in cells from lung tissue [33].
We also performed a gene-set enrichment analysis focusing on drugs, finding evidence of enrichment of TSA, which had been proposed to target several genes previously associated with asthma-related traits and drug metabolism, suggesting that TSA could be involved in the molecular mechanisms underlying the occurrence of asthma exacerbations despite ICS treatment. These findings demonstrate that GWAS approaches in combination with gene-set enrichment analyses seem to be a powerful strategy to explore potential novel therapeutic interventions, even in the absence of genome-wide associations [58, 59].
TSA is a hydroxamic acid extracted from the bacterial genus Streptomyces with a wide range of histone deacetylase (HDAC) inhibitor activities in mammalian cells [60]. Specifically, TSA belongs to a family of compounds acting on metal-dependent HDACs, inhibiting histone deacetylation and causing hyperacetylation of core histones, which is one of the major regulators of the chromatin structure [61]. Nonetheless, HDAC inhibitors have been demonstrated to act on diverse nonhistone substrates involved in several functions, such as cell signalling, chromatin structure and DNA repair, among others [62].
Interestingly, the potential clinical utility of HDAC inhibitors in asthma has been investigated [62]. Several studies in animal models [62–64] have suggested that the inhibition of HDACs by TSA could play an important role in the reduction of asthma development by decreasing airway inflammation and hyperresponsiveness [65]. These findings, together with evidence that HDACs regulate sensitivity to glucocorticosteroids [62], suggest that histone acetylation may play a key role in asthma development [66], and seems to be a promising target for alternatives to the standard medications currently used in the management of asthma. Specifically, in vivo experiments in allergen-challenged mice have demonstrated that treatment with TSA decreases eosinophils and lymphocytes levels in bronchial alveolar lavage. Reduced expression levels of inflammatory mediators such as Th2 cytokines were also detected [66]. Moreover, it has been found that TSA shows additive effects in combination with glucocorticosteroids, suggesting that it might target the main pathological processes in asthma through mechanisms of action different from the classical asthma anti-inflammatory medications [63]. Additionally, Banerjee et al. [63] found that TSA could have important functions in the inhibition of bronchoconstriction by inducing remodelling changes. It has been demonstrated that TSA treatment might inhibit the release of intracellular calcium, reducing ASM contraction in human lung slices and ASM cells in vitro expose to contractile agonists [63].
Although the effects of TSA on chromatin structure and regulation of gene expression in pulmonary tissues are still unclear [63], these findings suggest that TSA could potentially play an important role in asthma through epigenetic modifications and regulate the molecular mechanisms involved in response to ICS. Nonetheless, to the best of our knowledge, the effect of TSA on asthma patients has not been tested in clinical trials yet and little is known about the potential side-effects of this drug. For this reason, there is still a long way for the potential introduction of TSA as controller therapy in clinical practice.
The current study has some limitations that need to be acknowledged. First, the genome-wide significance level was reached neither in the discovery phase nor after combining the results with independent European studies. Although to our knowledge our study includes the largest sample size analysed in any GWAS of exacerbations despite ICS use performed in children and young adults with asthma to date, the lack of genome-wide associations could be explained by reduced statistical power given by differences in patient recruitment and definition of asthma exacerbations tested in association in both discovery and replication phases. Additionally, no covariates related to the aetiology of asthma exacerbations and exposure to potential environmental triggers were considered in the association analyses. Second, retrospective information about the occurrence or absence of asthma exacerbations partly based on self-reports was used, which could not be fully informative of the real ICS response. Moreover, a period of 6 or 12 months preceding the study enrolment was considered, which could have introduced substantial heterogeneity in the interpretation of treatment response, since more exacerbations are possible in additional 6 months and nonresponse might be more likely to occur in 12 months. Third, although the standard definition of severe asthma exacerbations established by the European Respiratory Society and the American Thoracic Society considering them as the need for unscheduled medical care because of asthma [16] was used, this information was incomplete for some of the European studies included in the discovery or replication phases. Therefore, data regarding unscheduled visits to general practitioners or respiratory disease specialists and school absences due to asthma were considered instead, which captures moderate asthma exacerbations. Additionally, no variables indicating whether ICS therapy had been initiated before or after exacerbations episodes were available. Altogether, this heterogeneity in data availability could represent a potential interpretation bias in terms of response to asthma treatment. Fourth, specific ICS dose and type or any index of treatment adherence were not included as covariates in the association analyses, since information related to these variables was not available for most of the studies included in this GWAS. Fifth, although in silico evaluation of the functional implication of CACNA2D3 and WNT5A on asthma exacerbations was carried out, in vitro experiments, pharmacogenomic research of pre-existing randomised controlled trials, and longitudinal asthma studies are needed to confirm their role in asthma treatment response.
In summary, our GWAS of asthma exacerbations in children and young adults treated with ICS revealed a novel association in Europeans. We also found evidence of replication of variants previously associated with different definitions of ICS response in asthma patients of European descent and suggested TSA as a potential novel therapy that could be implicated in mechanisms controlling asthma symptoms and moderate-to-severe exacerbations in patients treated with ICS. These findings suggest that the integration of different analytical methods could be a powerful strategy providing new insights into the molecular mechanisms underlying ICS response and suggesting alternative asthma therapies.
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Acknowledgements
The authors acknowledge the patients, families, recruiters, healthcare providers and community clinics for their participation in all the studies included in the PiCA consortium (http://pica-consortium.org). The authors thank the contribution of Teide High-Performance Computing facilities (http://teidehpc.iter.es) provided by the Instituto Tecnológico y de Energías Renovables (ITER, S.A.) to the results of this research and also the Centro Nacional de Genotipado-Plataforma de Recursos Biomoleculares-Instituto de Salud Carlos III (CeGen-PRB3-ISCIII; www.cegen.org) for the genotyping services provided. We acknowledge all the families who took part in ALSPAC, the midwives for their help in recruiting them, and the whole ALSPAC team, which includes interviewers, computer and laboratory technicians, clerical workers, research scientists, volunteers, managers, receptionists and nurses. The GALA II and SAGE studies collaborators include Shannon Thyne (University of California Los Angeles ,Los Angeles, CA, USA), Harold J. Farber (Baylor College of Medicine and Texas Children's Hospital, Houston, TX, USA), Denise Serebrisky (Jacobi Medical Center, New York, NY, USA), Rajesh Kumar (The Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, IL, USA), Emerita Brigino-Buenaventura (Kaiser Permanente, Vallejo, CA, USA), Michael A. LeNoir (Bay Area Pediatrics, Oakland, CA, USA), Kelley Meade (Children's Hospital and Research Center Oakland, Oakland, CA), William Rodriguez-Cintron (Veterans Caribbean Health Care System, San Juan, Puerto Rico), Pedro C. Avila (Northwestern University, Evanston, IL, USA), Jose R. Rodriguez-Santana (Centro de Neumologia Pediatrica, San Juan), Luisa N. Borrell (City University of New York, New York, NY) Adam Davis (Children's Hospital and Research Center Oakland), Saunak Sen (University of Tennessee, Knoxville, TN, USA) and Fred Lurmann (Sonoma Technologies, Petaluma, CA, USA). The authors acknowledge the families and patients for their participation and thank the numerous healthcare providers and community clinics for their support and participation in the GALA II and SAGE studies. In particular, the authors thank study coordinator Sandra Salazar (University of California San Francisco, San Francisco, CA, USA); the recruiters who obtained the data: Duanny Alva, Gaby Ayala-Rodriguez, Lisa Caine, Elizabeth Castellanos, Jaime Colon, Denise DeJesus, Blanca Lopez, Brenda Lopez, Louis Martos, Vivian Medina, Juana Olivo, Mario Peralta, Esther Pomares, Jihan Quraishi, Johanna Rodriguez, Shahdad Saeedi, Dean Soto and Ana Taveras
Footnotes
This article has supplementary material available from erj.ersjournals.com
Conflict of interest: N. Hernandez-Pacheco reports grants from Instituto de Salud Carlos III (ISCIII, FI16/00136) and co-funded by the European Social Funds from the European Union (ESF) “ESF invests in your future”, during the conduct of the study.
Conflict of interest: S.J. Vijverberg has nothing to disclose.
Conflict of interest: E. Herrera-Luis reports grants from the Spanish Ministry of Science, Innovation, and Universities (PRE2018-083837), during the conduct of the study.
Conflict of interest: J. Li has nothing to disclose.
Conflict of interest: Y.Y. Sio has nothing to disclose.
Conflict of interest: R. Granell has nothing to disclose.
Conflict of interest: A. Corrales has nothing to disclose.
Conflict of interest: C. Maroteau has nothing to disclose.
Conflict of interest: R. Lethem has nothing to disclose.
Conflict of interest: J. Perez-Garcia has nothing to disclose.
Conflict of interest: N. Farzan has nothing to disclose.
Conflict of interest: K. Repnik has nothing to disclose.
Conflict of interest: M. Gorenjak has nothing to disclose.
Conflict of interest: P. Soares has nothing to disclose.
Conflict of interest: L. Karimi has nothing to disclose.
Conflict of interest: M. Schieck has nothing to disclose.
Conflict of interest: L. Pérez-Méndez has nothing to disclose.
Conflict of interest: V. Berce has nothing to disclose.
Conflict of interest: R. Tavendale has nothing to disclose.
Conflict of interest: C. Eng has nothing to disclose.
Conflict of interest: O. Sardon has nothing to disclose.
Conflict of interest: I. Kull has nothing to disclose.
Conflict of interest: S. Mukhopadhyay reports grants from The Gannochy Trust, Perth and Kinross City Council and Scottish Enterprises Tayside, during the conduct of the study.
Conflict of interest: M. Pirmohamed reports grants from UK Department of Health and UK Medical Research Council, during the conduct of the study; grants from MRC Clinical Pharmacology Training Scheme (joint funding by MRC and Roche, UCB, Eli Lilly and Novartis), Joint PhD studentship funded by EPSRC and Astra Zeneca and grants from Bristol Myers Squibb, outside the submitted work.
Conflict of interest: K.M.C. Verhamme reports grants from ZonMw, during the conduct of the study; and works for a department who in the past received unconditional research grants from Yamanouchi, Pfizer/Boehringer Ingelheim, Novartis and GSK.
Conflict of interest: E.G. Burchard reports grants from the National Heart, Lung, and Blood Institute (NHLBI) of the US National Institutes of Health (NIH) (X01HL134589, X01HL134589,R01HL128439, R01HL135156, R01HL141992 and R01HL141845), the National Institute of Environmental Health Sciences (NIEHS) (R01ES015794 and R21ES24844), the National Institute on Minority Health and Health Disparities (NIMHD) (P60MD006902, R01MD010443 and R56MD013312), the National Institute of General Medical Sciences (NIGMS) (RL5GM118984), the Tobacco-Related Disease Research Program (award numbers 24RT-0025 and 27IR-0030), the National Human Genome Research Institute (NHGRI) (U01HG009080), the Sandler Family Foundation, the American Asthma Foundation, the Amos Medical Faculty Development Program from the Robert Wood Johnson Foundation, the Harry Wm. and Diana V. Hind Distinguished Professorship in Pharmaceutical Sciences II, during the conduct of the study.
Conflict of interest: M. Kabesch reports grants from European Union, German Ministry of Education and Research, German Research Foundation, during the conduct of the study; personal fees for consultancy from Bionorica, Sanofi, Novartis and Bencard, personal fees for lectures from ERS, EAACI, ATS, Novartis, Glaxo, Nutricia, Hipp and Allergopharma, outside the submitted work.
Conflict of interest: D.B. Hawcutt has nothing to disclose.
Conflict of interest: E. Melén has nothing to disclose.
Conflict of interest: U. Potočnik reports grants from Slovenian Research Agency (P3-0067) and Ministry of Education, Science and Sport Slovenia (MIZS) (SysPharmPediA grant C3330-16-500106), during the conduct of the study.
Conflict of interest: F.T. Chew reports grants from Singapore Ministry of Education Academic Research Fund, Singapore Immunology Network, National Medical Research Council (NMRC) (Singapore), Biomedical Research Council (BMRC) (Singapore), and the Agency for Science Technology and Research (A*STAR) (Singapore), during the conduct of the study; and consulting fees from Sime Darby Technology Centre, First Resources Ltd, Genting Plantation and Olam International, outside the submitted work.
Conflict of interest: K.G. Tantisira reports grants from U.S. National Institutes of Health, during the conduct of the study.
Conflict of interest: S. Turner has nothing to disclose.
Conflict of interest: C.M. Palmer has nothing to disclose.
Conflict of interest: C. Flores has nothing to disclose.
Conflict of interest: M. Pino-Yanes reports grants from Spanish Ministry of Economy, Industry and Competitiveness (funded by the Ramón y Cajal Program, RYC-2015-17205), and Instituto de Salud Carlos III (ISCIII) (funded by ISCIII through AES and EC within AAL framework, and the SysPharmPedia grant from the ERACoSysMed 1st Joint Transnational Call from the European Union under the Horizon 2020, AC15/00015), during the conduct of the study.
Conflict of interest: A.H. Maitland-van der Zee reports grants from GSK, during the conduct of the study; grants from Boehringer Ingelheim, personal fees for advisory board work from AstraZeneca and Boehringer Ingelheim, outside the submitted work.
Support statement: This study was supported by the awards (AC15/00015 and AC15/00058) funded by the Instituto de Salud Carlos III (ISCIII) through Strategic Action for Health Research (AES) and European Community (EC) within the Active and Assisted Living (AAL) Programme framework (M. Pino-Yanes, Olaia Sardón), the SysPharmPedia grant from the ERACoSysMed 1st Joint Transnational Call from the European Union under the Horizon 2020, and by the Spanish Ministry of Science, Innovation and Universities (grant SAF2017–83417R MICIU/AEI/FEDER, UE). The PACMAN study was funded by a strategic alliance between GlaxoSmithKline and Utrecht Institute for Pharmaceutical Sciences. The SLOVENIA study was financially supported by the Slovenian Research Agency (research core funding number P3-0067) and from SysPharmPedia grant, co-financed by Ministry of Education, Science and Sport Slovenia (MIZS) (contract number C3330-16-500106). GALA II was supported by the National Heart, Lung, and Blood Institute of the National Institute of Health (NIH) grants R01HL117004 and X01HL134589; study enrolment supported by the Sandler Family Foundation, the American Asthma Foundation, the RWJF Amos Medical Faculty Development Program, Harry Wm. and Diana V. Hind Distinguished Professor in Pharmaceutical Sciences II and the National Institute of Environmental Health Sciences grant R01ES015794. SAGE was funded by the National Heart, Lung, and Blood Institute of the National Institute of Health (NIH) grants R01HL117004 and X01HL134589; study enrolment supported by the Sandler Family Foundation, the American Asthma Foundation, the RWJF Amos Medical Faculty Development Program, Harry Wm. and Diana V. Hind Distinguished Professor in Pharmaceutical Sciences II. The SHARE Bioresource (GoSHARE) and SHARE have ongoing funding from NHS Research Scotland and established by funding from The Wellcome Trust Biomedical Resource (grant number 099177/Z/12/Z). Genotyping of samples from BREATHE-PAGES, GoSHARE, and SCSGES was carried out at CeGen-PRB3-ISCIII; supported by ISCIII and European Regional Development Fund (ERDF) (PT17/0019). ALSPAC was supported by the UK Medical Research Council and Wellcome (102215/2/13/2) and the University of Bristol. The Swedish Heart-Lung Foundation, the Swedish Research Council and Region Stockholm (ALF project and database maintenance) funded the BAMSE study. ESTATe was funded by an independent research grant by ZonMw project (113201006). The Childhood Asthma Management Program (CAMP) was supported by grants from NIH (R01HL127332 and R01NR013391). The PASS study was funded by the NHS Chair of Pharmacogenetics via the UK Department of Health. M. Pirmohamed is Emeritus NIHR Senior Investigator. Funding information for this article has been deposited with the Crossref Funder Registry.
- Received February 17, 2020.
- Accepted November 9, 2020.
- Copyright ©ERS 2021
References
