Asthma is a common chronic respiratory disease characterized by episodes of wheezing, coughing, shortness of breath, and airflow limitation, which vary over time and intensity.1 Moreover, about 75% of asthma patients also present atopy.1,2 Asthma is considered a complex disease with a multifactorial background, in which genetic factors are known to play a significant role, with heritability ranging between 35% and 95%.2 Among European countries, Spain exhibits variation in asthma prevalence, the Canary Islands having the highest prevalence (18%) compared to the rest of the Spanish regions (4% on average).3-5

While asthma symptoms may resolve spontaneously or in response to preventive medication, some patients with asthma may experience episodic flare-ups, known as exacerbations. The most widely used definition of asthma exacerbations comprises the administration of systemic corticosteroids, emergency room visits, or hospitalizations.6 These episodes not only negatively impact the patient's quality of life, long-term lung function, work productivity, and/or school attendance, but can also be life-threatening.1 Likewise, the associated socioeconomic burden extends to caregivers, the healthcare system, and the whole community.1

Among the asthma-associated genetic factors, the serpin family A member 1 gene (SERPINA1) participates in the development of several respiratory diseases, including asthma,7,8 and it is also associated with atopy in asthma patients.9SERPINA1 gene encodes alpha-1 antitrypsin (AAT), the most abundant antiprotease in the human serum,10 plays a fundamental role as a serine protease inhibitor, protecting the lower respiratory tract against neutrophil elastase, in addition to having other immunomodulatory and anti-inflammatory properties.11 When serum levels of AAT decrease below a protective threshold,12 reduced protection against elastase activity in the lung could lead to alveoli degradation.13

Different SERPINA1 gene variants are known to produce AAT deficiency (AATD), which is considered one of the most common hereditary disorders of European descent populations.14 AATD is diagnosed by measurement of AAT serum levels and characterization of AAT isoforms by protein isoelectric focusing (Pi*AAT phenotypes). Pi*M is considered the reference AAT variant, associated with normal AAT levels, while two canonical Pi*AAT variants, known as Pi*Z and Pi*S, have been classically associated with AATD.15Pi*Z variant is related to severe AATD,16 and is caused by a missense mutation (p.Glu342Lys, rs28929474) in the SERPINA1 gene, with a minor allele frequency (MAF) < 1%, reaching the highest MAF in European populations (2%). Pi*S variant is related to moderate AATD and is produced by another missense mutation of the SERPINA1 gene (p.Glu264Val, rs17580) with the highest MAF in European-descent populations (6%)17. Interestingly, the Spanish population shows the highest allele frequency for this polymorphism among the populations from the 1000 Genomes project (9.3%).18

To the best of our knowledge, the association of SERPINA1 classic genetic variants with asthma exacerbations has not been studied yet. In this sense, populations of the Canary Islands are interesting due to the elevated asthma prevalence in this region, and the unique genomic features found in the different islands.19,20 Indeed, novel defective SERPINA1 alleles have been characterized in La Palma island,21 where frequencies of Pi*S and Pi*Z alleles are higher than expected.22 In the present work, we performed the first genetic association study of SERPINA1 variants with asthma exacerbations, considering subjects with asthma from La Palma Island as a model. In addition, were attempted to replicate our findings in Canary Islanders, mainland Spanish subjects, and other European populations.

To the best of our knowledge, this is the first study examining the association of AATD and SERPINA1 variants with asthma exacerbations. A higher level of AAT was associated with a protective effect for asthma exacerbations among Canary Islanders from La Palma. In addition, AATD, considered when serum protein level was below 80 mg/dl, was associated with a higher risk for asthma exacerbations. In the same population, the rs17580 (Pi*S) and rs28929474 (Pi*Z) SERPINA1 variants were associated with a higher risk of asthma exacerbations. The rs28929474 signal was replicated in an independent sample of the Canary Islands and Finnish populations, but not in other Spanish or European populations.

Several genes have been associated with predisposition to this flare-up episode,29-36 but in-depth studies, carried out in additional populations, are necessary to identify the whole genetic component involved in asthma exacerbations. Nevertheless, AATD is considered an underdiagnosed disease, as only about 10% of patients are estimated to be properly diagnosed.37 Therefore, the scarce AATD diagnosis bias could underestimate its association with asthma. Since AAT is the main inhibitor of neutrophilic serine proteinase and acts as an important anti-inflammatory protein with pronounced immunomodulatory activity, the link between AATD and asthma might be represented by the elastase/antielastase imbalance caused by AATD. Alternatively, the proinflammatory effect caused by AAT serum levels reduction could also underlie the association found.7 Interestingly, a previous study observed that protein levels of AAT are elevated during asthma exacerbations,38 which agrees with our findings.

Despite the important role of SERPINA1 variants in COPD,27 the associations found were not driven by this condition, as they remained significant when all asthmatic patients with pulmonary obstruction were excluded from the analysis. Moreover, the association of AATD with asthma severity was also confirmed by analyzing asthma control values as an alternative outcome. Given that IgE and eosinophil levels are related to allergic asthma,39 and the high prevalence of atopy in the Canary Islands,5 we evaluated the association of SERPINA1 variants and genotypes with these phenotypes. However, no associations were found in this case, suggesting the SERPINA1 variants are not directly involved in the immunomodulation of the allergic response in these individuals. In previous studies, the Pi*Z variant has been associated with smoking behavior17, and the Pi*S variant has been mainly associated with the risk of developing COPD in smokers.17 However, our associations of Pi*S and Pi*Z alleles, AAT levels, and AATD with asthma exacerbations were not explained by smoking history, body mass index, or ICS use.

The associations found in the discovery population motivated us to assess the validation in Spanish subjects considering the overall population and a subset of individuals with two generations of Canary Islands origin. The latter comparison allowed us to evaluate the possible existence of a population-specific effect. It is known that populations from the Canary Islands present a recent admixture involving North African, European, and sub-Saharan African ancestry with variable ancestry, depending on the island.40 Although no relationship has been found between genetic ancestry and asthma when individuals from different islands of the Canary Archipelago were analyzed,41 studies based on island-specific populations are yet to be addressed. In this sense, evidence of validation was found for the association of rs28929474 (Pi*Z) with asthma exacerbations in Canary Islanders, but not in other Spanish populations.

The rs28929474 (Pi*Z) variant was also associated with asthma hospitalizations in the Finnish population, suggesting the possibility that a founder effect in certain isolated populations could cause an increase in SERPINA1 deficient alleles associated with the risk of asthma exacerbations. In this sense, the lack of replication in the independent populations from mainland Spain may be due to differences in allele frequencies among the discovery and replication populations. In previous studies,38,42 a high frequency of both polymorphisms was found among Spanish individuals, where the Pi*S and Pi*Z variants have a frequency of 10 and 2%, respectively.43 Interestingly, the frequency of the Pi*Z variant in our study was higher in La Palma (3.8%), followed by Canary Islanders from GEMAS (3.1%), and mainland Spaniards from MEGA (1.1%). In the case of the Finnish population, the frequency of Pi*Z was only two times lower than in La Palma (1.9%), but the frequency of Pi*S was about 10 times lower (0.8%), which could explain the lack of replication of the Pi*S variant in this population.

To the best of our knowledge, the present work represents the first evidence of the association of AATD and SERPINA1 classic variants with asthma exacerbations. Moreover, we have demonstrated the robustness of the results by including strict quality control procedures and considering multiple demographic and clinical confounders in the association analyses. Additionally, we have analyzed different populations, not only genotyped in our study but also from different public databases queries.

Nonetheless, some limitations must be considered. First, the Pi*S association with asthma exacerbations was not validated in all populations analyzed in the replication stage. Although the association found could be population specific, the lack of replication could also be due to the reduced statistical power derived from the smaller sample size in the non-Canary Islander replication set of samples. Second, different endotypes of asthma were not assessed and could underlie the different results obtained in the cohorts analyzed. Third, AAT serum levels were not available in GEMAS and MEGA. Therefore, it was not possible to confirm the association between AATD and asthma exacerbations in the replication phase. Fourth, other possible confounding factors were not evaluated, for example, atopy, rhinitis, or other individual or environmental conditions, since data was not available for all cohorts, and these uncontrolled conditions could mask the associations in the replication populations. Fifth, the recruitment of patients is heterogeneous between studies since CAATDPUL enrolled patients from the Pneumonology unit only, but GEMAS and MEGA enrolled patients from Pneumonology and Allergology units. Despite correcting the regression models by the recruitment center, this feature could be another reason for the lack of replication. Additionally, differences in the environment could also affect the associations through gene-environment interactions.

Sixth, rare variants can also cause AATD, and such type of variation was not assessed. Therefore, future sequencing efforts analyzing the whole spectrum of genetic variation in the SERPINA1 gene could reveal additional associations with asthma exacerbations.

Personalized medicine has shown the existence of drugs with application to specific populations.44 In this sense, it could be helpful to measure AAT serum levels and assess the SERPINA1 genetic variants in asthmatic patients routinely in clinical practice to detect carriers of SERPINA1 deficient variants, during which implementing AAT replacement therapy could be a potential treatment to reduce asthma exacerbations. This approach would especially be relevant to patients who do not respond to other asthma treatments, in order to decrease disease morbidity and mortality.

We describe for the first time the association of the SERPINA1 gene Pi*S (rs17580) and Pi*Z (rs28929474) variants, AAT levels, and AATD with asthma exacerbations in patients from La Palma (Canary Islands, Spain). In addition, the association of the Pi*Z allele, the most important SERPINA1 variant in clinical practice, was validated in another set of Canary Islanders and individuals from the Finnish population. This suggests AATD is a potential therapeutical target for treating asthma exacerbations in specific populations.

M.P.Y. and M.G.C. are the guarantors of the paper, taking responsibility for the integrity of the work, from inception to the published article. E.M.G. was responsible for formal analysis and writing the original draft. J.M.H.P., M.P.Y., and M.G.C. contributed to the conceptualization, and M.P.Y. and M.G.C. also contributed to methodology and supervision. Resources were carried out by J.M.H.P., J.A.P.P., R.G.P., E.M.L., I.S.M., O.S., P.C., P.P.G., J.K.M., V.d.P., F.L.D., J.V., and M.P.Y. All the authors support with the investigation, writing-review, and editing. Funding acquisition was carried out by J.M.H.P. and M.P.Y.

This study was funded by the Spanish Ministry of Science and Innovation MCIN/AEI/10.13039/501100011033 (PID2020–116274RB-I00) and by Sociedad Española de Neumología y Cirugía Torácica (SEPAR http://www.separ.es), grant number 1264–2022. Authors declare they have no competing interests or other interests that might be perceived to influence the interpretation of the manuscript. No supporting institution may gain or lose financially through this publication. Genotyping of samples from GEMAS and MEGA studies was funded by the Spanish Ministry of Science and Innovation (SAF2017–87417R) at the Spanish National Cancer Research centre, in the Human Genotyping lab, a member of CeGen, PRB3, and was supported by grant PT17/0019, of the PE I + D + i 2013–2016, funded by Instituto de Salud Carlos III (ISCIII) and European Regional Development Fund. Genotyping of GEMAS was also partially funded by Fundación Canaria Instituto de Investigación Sanitaria de Canarias (PIFIISC19/17). E.M.G. was supported by a fellowship (TESIS2022010045) awarded by the Board of Economy, Industry, Trade, and Knowledge of the Canary Islands Government, with a European Social Fund co-financing rate managed by the Canary Islands Agency for Research, Innovation, Society, and Information (ACIISI). J.P.G. was funded by an FPU fellowship (FPU19/02,175) granted by the Spanish Ministry of Universities. E.H.L. was supported by a fellowship awarded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future” (PRE2018–083,837). V.P. has received grant support from MSD. V.d.P. was supported by CIBER de Enfermedades Respiratorias, ISCIII, Spain. M.P.Y. was funded by the Ramón y Cajal Program (RYC-2015–17,205) by MCIN/AEI/10.13039/501100011033 and by the European Social Fund “ESF Investing in your future”. M.P.Y. and J.V. were supported by CIBER de Enfermedades Respiratorias, ISCIII, Spain (CB/06/06/1088).

The sponsor had no role in the design of the study, the collection, and analysis of the data, or the preparation of the manuscript.

The authors thank the patients, families, recruiters, healthcare providers, and community clinics for their participation in the studies analyzed in this manuscript. The genotyping service was carried out at CEGEN-PRB3-ISCIII; it is supported by grant PT17/0019, of the PE I + D + i 2013–2016, funded by ISCIII and ERDF.

This study was funded by the Spanish Ministry of Science and Innovation MCIN/AEI/10.13039/501100011033 (PID2020–116274RB-I00) and by Sociedad Española de Neumología y Cirugía Torácica (SEPAR http://www.separ.es), grant number 1264–2022.