A meta-analysis of genome-wide association studies of childhood wheezing phenotypes identifies ANXA1 as a susceptibility locus for persistent wheezing
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Genome-wide association studies on asthma have been challenging due to innate heterogeneity and the syndromic nature of asthma, variable accuracy in phenotyping, and potential gene-environment interactions. Here, the authors identified genetic loci associated with subtypes of childhood wheezing in combined data of multiple birth cohorts, by coupling latent class analysis of clinical phenotypic data with GWAS discovery. A mechanistically plausible genetic locus close to annexin 1 (ANXA1) was associated exclusively with early-onset persistent wheeze and provides new translatable molecular insight into asthma pathogenesis.
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Abstract
Many genes associated with asthma explain only a fraction of its heritability. Most genome-wide association studies (GWASs) used a broad definition of ‘doctor-diagnosed asthma’, thereby diluting genetic signals by not considering asthma heterogeneity. The objective of our study was to identify genetic associates of childhood wheezing phenotypes.
Methods:
We conducted a novel multivariate GWAS meta-analysis of wheezing phenotypes jointly derived using unbiased analysis of data collected from birth to 18 years in 9568 individuals from five UK birth cohorts.
Results:
Forty-four independent SNPs were associated with early-onset persistent, 25 with pre-school remitting, 33 with mid-childhood remitting, and 32 with late-onset wheeze. We identified a novel locus on chr9q21.13 (close to annexin 1 [ ANXA1 ], p<6.7 × 10 -9 ), associated exclusively with early-onset persistent wheeze. We identified rs75260654 as the most likely causative single nucleotide polymorphism (SNP) using Promoter Capture Hi-C loops, and then showed that the risk allele (T) confers a reduction in ANXA1 expression. Finally, in a murine model of house dust mite (HDM)-induced allergic airway disease, we demonstrated that anxa1 protein expression increased and anxa1 mRNA was significantly induced in lung tissue following HDM exposure. Using anxa1 -/- deficient mice, we showed that loss of anxa1 results in heightened airway hyperreactivity and Th2 inflammation upon allergen challenge.
Conclusions:
Targeting this pathway in persistent disease may represent an exciting therapeutic prospect.
Funding:
UK Medical Research Council Programme Grant MR/S025340/1 and the Wellcome Trust Strategic Award (108818/15/Z) provided most of the funding for this study.
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Author Response
Reviewer #2 (Public Review):
Granell et al. investigated genetic factors underlying wheezing from birth to young adulthood using a robust data-driven approach with the aim of understanding the genetic architecture of different wheezing phenotypes. The association of 8.1 million single nucleotide polymorphisms (SNPs) with wheeze phenotypes derived from birth to 18 years of age was evaluated in 9,568 subjects from five independent cohorts from the United Kingdom. This meta-genome-wide association study (GWAS) revealed the suggestive association of 134 independent SNPs with at least one wheezing subtype. Among these, 85 genetic variants were found to be potentially causative. Indeed, some of these were located nearby well-known asthma loci (e.g., the 17q21 chromosome band), although ANXA1 was revealed for the first time …
Author Response
Reviewer #2 (Public Review):
Granell et al. investigated genetic factors underlying wheezing from birth to young adulthood using a robust data-driven approach with the aim of understanding the genetic architecture of different wheezing phenotypes. The association of 8.1 million single nucleotide polymorphisms (SNPs) with wheeze phenotypes derived from birth to 18 years of age was evaluated in 9,568 subjects from five independent cohorts from the United Kingdom. This meta-genome-wide association study (GWAS) revealed the suggestive association of 134 independent SNPs with at least one wheezing subtype. Among these, 85 genetic variants were found to be potentially causative. Indeed, some of these were located nearby well-known asthma loci (e.g., the 17q21 chromosome band), although ANXA1 was revealed for the first time to play an important role in early-onset persistent wheezing. This was strongly supported by functional evidence. One of the top ANXA1 SNPs associated with wheezing was found to be potentially involved in the regulation of the transcription of this gene due to its location at the promoter region. This polymorphism (rs75260654) had been previously evidenced to regulate the ANXA1 expression in immune cells, as well as in pulmonary cells through its association as an eQTL. Protein-protein network analyses revealed the interaction of ANXA1 with proteins involved in asthma pathophysiology and regulation of the inflammatory response. Additionally, the authors conducted a murine model, finding increased anxa1 levels after a challenge with house dust mite allergens. Mice deficient in anxa1 showed decreased lung function, increased eosinophilia, and Th2 cell levels after allergen stimulation. These results suggest the dysregulation of the immune response in the lungs, eosinophilia, and Th2-driven exacerbations in response to allergens as a result of decreased levels of anxa1. This coincides with evidence of lower plasmatic ANXA1 levels in patients with uncontrolled asthma, suggesting this locus is a very promising candidate as a target of novel therapeutic strategies.
Limitations of this piece of work that need to be acknowledged:
(1) the manual and visual inspection of Locus Zoom plots for the refinement of association signals and identification of functional elements does not seem to be objective enough;
This is an important observation and we have now added the following text in the Discussion which can be found on lines 400-2 Revised Main Manuscript:
“Finally, the manual and visual inspection of Locus Zoom plots for the refinement of association signals and identification of functional elements was an objective approach which might have undermined the findings.“
(2) the sample size is limited, although the statistical power was improved by the assessment of very accurate disease sub-phenotype;
This point was already mentioned as a limitation and it can now be found in lines 349-365 Revised Main Manuscript:
“By GWAS standards, our study is comparatively small and may be considered to be underpowered. The sample size may be an issue when using an aggregated definition (such as “doctor-diagnosed asthma”) but is less likely to be an issue when primary outcome is determined by deep phenotyping. This is indirectly confirmed in our analyses. Our primary outcome was derived through careful phenotyping over a period of more than two decades in five independent birth cohorts, and although comparatively smaller than some asthma GWASs, our study proved to be powered enough to detect previously identified key associations (e.g. chr17q21 locus). Precise phenotyping has the potential to identify new risk loci. For example, a comparatively small GWAS (1,173 cases and 2,522 controls) which used a specific subtype of early-onset childhood asthma with recurrent severe exacerbations as an outcome, identified a functional variant in a novel susceptibility gene CDHR3 (SNP rs6967330) as an associate of this disease subtype, but not of doctor-diagnosed asthma(51). This important discovery was made with a considerably smaller sample size but using a more precise asthma subtype. In contrast, the largest asthma GWAS to date had a ~40-fold higher sample size(7), but reported no significant association between CDHR3 and aggregated asthma diagnosis. Therefore, with careful phenotyping, smaller sample sizes may be adequately powered to identify larger effect sizes than those in large GWASs with broader outcome definitions(52).”
(3) association signals with moderate significance levels but with strong functional evidence were found;
We do not think of this as a limitation but as a strength. We were able to support our genetic results with evidence from experimental mouse models.
(4) no direct replication of the findings in independent populations including diverse ancestry groups was described.
This point was already mentioned as a limitation and it can now be found in lines 375-391 and 392-399 Revised Main Manuscript.
“We are cognisant that there may be a perception of the lack of replication of our GWAS findings. We would argue that direct replication is almost certainly not possible in other cohorts, as phenotypes for replication studies should be homogenous(56). However, there is a considerable heterogeneity in LCA-derived wheeze phenotypes between studies, and although phenotypes in different studies are usually designated with the same names, they differ between studies in temporal trajectories, distributions within a population, and associated risk factors(57). This heterogeneity is in part consequent on the number and the non-uniformity of the timepoints used, and is likely one of the factors responsible for the lack of consistent associations of discovered phenotypes with risk factors reported in previous studies(58). This will also adversely impact the ability to identify phenotype-specific genetic associates. For example, we have previously shown that less distinct wheeze phenotypes in PIAMA were identified compared to those derived in ALSPAC(59). Thus, phenotypes that are homogeneous to those in our study almost certainly cannot readily be derived in available populations. This is exemplified in our attempted replication of ANXA1 findings in PIAMA cohort (see OLS, Table E12). In this analysis, the number of individuals assigned to persistent wheezing in PIAMA was small (40), associates of this phenotype differed to those in STELAR cohorts, and the SNPs’ imputation scores were low (<0.60), which meant the conditions for replication were not met.”
“Our study population is of European descent, and we cannot generalize the results to different ethnicities or environments. It is important to highlight the under-representation of ethnically diverse populations in most GWASs(9). To mitigate against this, large consortia have been formed, which combine the results of multiple ethnically diverse GWASs to increase the overall power to identify asthma-susceptibility loci. Examples include the GABRIEL(6), EVE(60) and TAGC(7) consortia, and the value of diverse, multi-ethnic participants in large-scale genomic studies has recently been shown(61). However, such consortia do not have the depth of longitudinal data to allow the type of analyses which we carried out to derive a multivariable primary outcome.”
Nonetheless, the robustness and consistency of the findings supported by different analytical and experimental layers is the major strength of this study.
The authors successfully achieved the aims of the study, strongly supported by the results presented. This study not only provides an exciting novel locus for wheezing with potential implications in the development of alternative therapeutic strategies but also opens the path for better-powered research of asthma genetics, focused on accurate disease phenotypes derived by innovative data-driven approaches that might speed up the process to disentangle the missing heritability of asthma, making use of still useful GWAS approaches.
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eLife assessment
Genome-wide association studies on asthma have been challenging due to innate heterogeneity and the syndromic nature of asthma, variable accuracy in phenotyping, and potential gene-environment interactions. Here, the authors identified genetic loci associated with subtypes of childhood wheezing in combined data of multiple birth cohorts, by coupling latent class analysis of clinical phenotypic data with GWAS discovery. A mechanistically plausible genetic locus close to annexin 1 (ANXA1) was associated exclusively with early-onset persistent wheeze and provides new translatable molecular insight into asthma pathogenesis.
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Reviewer #1 (Public Review):
Asthma is a syndromic disease with multiple subtypes with different pathogenetic paths to a final wheezing phenotype. This limits the insights gleaned from genetic investigations of asthma. One of the most important phenotypes is early life onset wheezing, which persists. Here, the authors use data from multiple birth cohorts and by coupling latent class analysis of clinical phenotypic data with GWAS discovery, identify a novel locus close to annexin 1 (ANXA1) associated exclusively with early-onset persistent wheeze. The methodology is a major strength of the work and highlights the importance of acquiring and analysing phenotypic over simple use of doctor labels for complex diseases.
The authors went on to demonstrate a putative mechanism such that the risk allele (T) may confer a reduction in ANXA1 …
Reviewer #1 (Public Review):
Asthma is a syndromic disease with multiple subtypes with different pathogenetic paths to a final wheezing phenotype. This limits the insights gleaned from genetic investigations of asthma. One of the most important phenotypes is early life onset wheezing, which persists. Here, the authors use data from multiple birth cohorts and by coupling latent class analysis of clinical phenotypic data with GWAS discovery, identify a novel locus close to annexin 1 (ANXA1) associated exclusively with early-onset persistent wheeze. The methodology is a major strength of the work and highlights the importance of acquiring and analysing phenotypic over simple use of doctor labels for complex diseases.
The authors went on to demonstrate a putative mechanism such that the risk allele (T) may confer a reduction in ANXA1 expression. Altered ANXA1 expression was additionally recapitulated in a murine model of house dust mite (HDM)-induced allergic airway disease. In this model, ANXA1 increased, rather than decreased, which may be attributable to its role in resolving inflammation. ANXA1-deficient mice had a more severe phenotype. This strengthens the evidence for causality in the novel link between ANXA1 and asthma and opens the door for further investigations. While novel for this link, the finding is well supported by prior knowledge about ANXA1-related pathways and inflammation. ANXA1 is known to participate in phospholipase A2-dependent reduction of inflammatory mediator production. Glucocorticoids increase ANXA1 levels. ANXA1 deficiency leads to airway hyperreactivity in mice. Overall, ANXA1 appears to be suitable as a therapeutic target and this may spur further investigations into the pathway.
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Reviewer #2 (Public Review):
Granell et al. investigated genetic factors underlying wheezing from birth to young adulthood using a robust data-driven approach with the aim of understanding the genetic architecture of different wheezing phenotypes. The association of 8.1 million single nucleotide polymorphisms (SNPs) with wheeze phenotypes derived from birth to 18 years of age was evaluated in 9,568 subjects from five independent cohorts from the United Kingdom. This meta-genome-wide association study (GWAS) revealed the suggestive association of 134 independent SNPs with at least one wheezing subtype. Among these, 85 genetic variants were found to be potentially causative. Indeed, some of these were located nearby well-known asthma loci (e.g., the 17q21 chromosome band), although ANXA1 was revealed for the first time to play an …
Reviewer #2 (Public Review):
Granell et al. investigated genetic factors underlying wheezing from birth to young adulthood using a robust data-driven approach with the aim of understanding the genetic architecture of different wheezing phenotypes. The association of 8.1 million single nucleotide polymorphisms (SNPs) with wheeze phenotypes derived from birth to 18 years of age was evaluated in 9,568 subjects from five independent cohorts from the United Kingdom. This meta-genome-wide association study (GWAS) revealed the suggestive association of 134 independent SNPs with at least one wheezing subtype. Among these, 85 genetic variants were found to be potentially causative. Indeed, some of these were located nearby well-known asthma loci (e.g., the 17q21 chromosome band), although ANXA1 was revealed for the first time to play an important role in early-onset persistent wheezing. This was strongly supported by functional evidence. One of the top ANXA1 SNPs associated with wheezing was found to be potentially involved in the regulation of the transcription of this gene due to its location at the promoter region. This polymorphism (rs75260654) had been previously evidenced to regulate the ANXA1 expression in immune cells, as well as in pulmonary cells through its association as an eQTL. Protein-protein network analyses revealed the interaction of ANXA1 with proteins involved in asthma pathophysiology and regulation of the inflammatory response. Additionally, the authors conducted a murine model, finding increased anxa1 levels after a challenge with house dust mite allergens. Mice deficient in anxa1 showed decreased lung function, increased eosinophilia, and Th2 cell levels after allergen stimulation. These results suggest the dysregulation of the immune response in the lungs, eosinophilia, and Th2-driven exacerbations in response to allergens as a result of decreased levels of anxa1. This coincides with evidence of lower plasmatic ANXA1 levels in patients with uncontrolled asthma, suggesting this locus is a very promising candidate as a target of novel therapeutic strategies.
Limitations of this piece of work that need to be acknowledged: (1) the manual and visual inspection of Locus Zoom plots for the refinement of association signals and identification of functional elements does not seem to be objective enough; (2) the sample size is limited, although the statistical power was improved by the assessment of very accurate disease sub-phenotype; (3) association signals with moderate significance levels but with strong functional evidence were found; (4) no direct replication of the findings in independent populations including diverse ancestry groups was described. Nonetheless, the robustness and consistency of the findings supported by different analytical and experimental layers is the major strength of this study.
The authors successfully achieved the aims of the study, strongly supported by the results presented. This study not only provides an exciting novel locus for wheezing with potential implications in the development of alternative therapeutic strategies but also opens the path for better-powered research of asthma genetics, focused on accurate disease phenotypes derived by innovative data-driven approaches that might speed up the process to disentangle the missing heritability of asthma, making use of still useful GWAS approaches.
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Reviewer #3 (Public Review):
Genome-wide association studies on asthma have been challenging, due to the accuracy of phenotyping and potential gene-environment interactions. Thus, the authors aimed to identify genetic loci associated with subtypes of childhood wheezing. One main strength of this investigation is that the data availability of wheeze from birth to adolescence among multiple birth cohorts allowed the sub-phenotyping on a large scale and high statistical power. The study is properly designed and the conclusions are well-supported. Understanding the heterogeneity in subtypes of childhood wheezing is of great clinical interest and may help inform future directions in disease prediction and prevention.
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