PLOS ONE RESEARCH ARTICLE Association of the CFTR gene with asthma and airway mucus hypersecretion Astrid Crespo-Lessmann ID1*, Sara Bernal2, Elisabeth del Rı́o2, Ester Rojas2, Carlos Martı́nez-Rivera3, Nuria Marina4, Abel Pallarés-Sanmartı́n5, Silvia Pascual6, Juan Luis Garcı́a-Rivero7, Alicia Padilla-Galo8, Elena Curto1, Carolina Cisneros9, José Serrano10, Montserrat Baiget2, Vicente Plaza1, Emerging Asthma Group¶ 1 Department of Respiratory Medicine, Hospital de la Santa Creu i Sant Pau, Institute of Sant Pau Biomedical Research (IBB Sant Pau), Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Department of Genetics, Hospital de la Santa Creu i Sant Pau, Institute of Sant Pau Biomedical Research (IBB Sant Pau), Centro de Investigación a1111111111 Biomédica en Red de Enfermedades Raras (CIBERER, U705), Barcelona, Spain, 3 Department of a1111111111 Respiratory Medicine, H. German Trias i Pujol, CIBERES, Badalona, Spain, 4 Department of Respiratory a1111111111 Medicine, H. de Cruces, Barakaldo, Vizcaya, Spain, 5 Department of Respiratory Medicine, H. Álvaro a1111111111 Cunqueiro, Vigo, Spain, 6 Department of Respiratory Medicine, H. de Galdakao, Vizcaya, Spain, 7 Department of Respiratory Medicine, H. Laredo, Cantabria, Spain, 8 Department of Respiratory Medicine, a1111111111 H. Costa del Sol de Marbella, Málaga, Spain, 9 Department of Respiratory Medicine, H. U. de La Princesa, Madrid, Spain, 10 Department of Respiratory Medicine, Hospital Comarcal de Inca, Baleares, Spain ¶ Membership of the Emerging Asthma Group is Provided in the Acknowledgments. * acrespo@santpau.cat OPEN ACCESS Citation: Crespo-Lessmann A, Bernal S, del Rı́o E, Rojas E, Martı́nez-Rivera C, Marina N, et al. (2021) Abstract Association of the CFTR gene with asthma and airway mucus hypersecretion. PLoS ONE 16(6): e0251881. https://doi.org/10.1371/journal. pone.0251881 Introduction Asthma with airway mucus hypersecretion is an inadequately characterized variant of Editor: Ritesh Agarwal, Postgraduate Institute of Medical Education and Research, INDIA asthma. While several studies have reported that hypersecreting patients may carry genetic variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, many of Received: December 2, 2020 those studies have been questioned for their numerous limitations and contradictory results. Accepted: May 5, 2021 Published: June 4, 2021 Objectives Peer Review History: PLOS recognizes the (1) To determine the presence of genetic variants of the CFTR gene in patients with asthma benefits of transparency in the peer review process; therefore, we enable the publication of with and without airway mucus hypersecretion. (2) To identify the clinical, inflammatory and all of the content of peer review and author functional characteristics of the asthma phenotype with airway mucus hypersecretion. responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0251881 Method Copyright: © 2021 Crespo-Lessmann et al. This is Comparative multicentre cross-sectional descriptive study that included 100 patients with an open access article distributed under the terms asthma (39 hypersecretors and 61 non-hypersecretors). Asthmatic hypersecretion was of the Creative Commons Attribution License, defined as the presence of cough productive of sputum on most days for at least 3 months which permits unrestricted use, distribution, and in 2 successive years. The patients were tested for fractional exhaled nitric oxide, spirome- reproduction in any medium, provided the original author and source are credited. try, induced sputum cell count, total immunoglobulin E (IgE), peripheral blood eosinophil count, C-reactive protein, blood fibrinogen and blood albumin and underwent a skin prick Data Availability Statement: All relevant data are within the paper and its Supporting Information test. Asthma control and quality of life were assessed by the Asthma Control Test and Mini files. Asthma Quality of Life questionnaires, respectively. Blood DNA samples were collected PLOS ONE | https://doi.org/10.1371/journal.pone.0251881 June 4, 2021 1 / 13 PLOS ONE CFTR gene, asthma and airway mucus hypersecretion Funding: Grant from de Spanish Society of from the patients and next-generation sequencing using a MiSeq sequencer and the Illu- Pulmonology and Thoracic Surgery (SEPAR) to Dr. mina platform was used for the CFTR gene analysis. Astrid Crespo Lessmann, and Astra Zeneca. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of Results the manuscript. Genetic differences were observed in the c.1680-870T>A polymorphism of the CFTR gene, Competing interests: I have read the journal’s significantly more evident in hypersecretors than in non-hypersecretors: 78.94% vs. 59.32% policy and the authors of this manuscript have the in the majority allele and 21.05% vs. 40.67% in the minority allele (p = 0.036). Clinically, following competing interests: AC has received fees in the last three years for talks at meetings asthma hypersecretors compared to non-hypersecretors were older (57.4 years vs. 49.4 sponsored by Chiesi, Esteve Laboratories, years; p = 0.004); had greater asthma severity (58.9% vs. 23.7%; p = 0.005); experienced GlaxoSmithKline, Novartis, Ferrer, Zambón and greater airway obstruction (FEV1/FVC% 64.3 vs. 69.5; p = 0.041); had poorer asthma con- Boehringer Ingelheim, has received travel and trol (60% vs. 29%; p = 0.021); had lower IgE levels (126.4 IU/mL vs. 407.6 IU/mL; p = attendance expenses for conferences from Novartis, Bial, Teva and FAES Farma and has 0.003); and were less likely to have a positive prick test (37.5% vs. 68.85%; p = 0.011). received funds/grants for research projects from several state agencies and non-profit foundations Conclusion and from AstraZeneca. EC reports non-financial support from Astrazeneca, personal fees from The results suggest that patients with asthma and with mucus hypersecretion (1) may have Boehringer-Ingleheim, personal fees and non- a different phenotype and disease mechanism produced by an intronic polymorphism in the financial support from Chiesi, non-financial support CFTR gene (NM_000492.3:c.1680-870T>A), and (2) may have a poorer clinical outcome from Novartis, non-financial support from characterized by severe disease and poorer asthma control with a non-allergic inflammatory Menarini, non-financial support from ALK, outside the submitted work. SB, EdR, ER, CM, NM, AP, SP, phenotype. JG, AP, CC, JS and MB have no conflicts of interest to declare. VP has received fees in the last three years for talks at meetings sponsored by AstraZeneca, Boehringer-Ingelheim, MSD and Chiesi, has received travel and attendance Introduction expenses for conferences from AstraZeneca, Chiesi In clinical practice, it is common to encounter patients with severe asthma who only partially and Novartis, has acted as a consultant for ALK, respond to steroid treatment and who have marked airway mucus hypersecretion. The AstraZeneca, Boehringer, MSD, MundiPharma and increase in mucus secretion may be mild or severe, marked only by chronic bronchitis or by Sanofi, and has received funds/grants for research projects from several state agencies and non-profit recurring bronchial infections, respectively; in extreme cases, patients may develop infectious foundations and from AstraZeneca, Chiesi and bronchiectasis and bronchiolitis. Probably because ‘airway mucus secretion’ is not usually Menarini. This does not alter our adherence to included in cluster analyses of asthma, this condition is not recognized as a specific phenotype. PLOS ONE policies on sharing data and materials. Wheaterall et al [1] evaluated patients with airflow obstruction and airway mucus hyperse- cretion, finding a poorer response to glucocorticoid treatment and more frequent hospital admissions due to exacerbations in patients with airway mucus hypersecretion secondary to chronic obstructive pulmonary disease (COPD) or asthma (bronchial hyperresponsiveness, ele- vated fractional exhaled nitric oxide (FeNO), rhinitis, dermatitis and blood eosinophilia). Muta- tions or polymorphisms in the cystic fibrosis transmembrane conductance regulator (CFTR) gene were detected in another study of 4 hypersecreting patients with asthma, with a neutro- philic inflammatory phenotype, bronchiectasis, pansinusitis and respiratory infections [2]; this would suggest that a possible explanation for airway mucus hypersecretion may be that having asthma results in a combination of asthma and an attenuated form of cystic fibrosis (CF) in patients with a genetic alteration in the CFTR gene. By the 1990s, several studies had confirmed that carriers of a mutation for CF were at an increased risk of asthma and of greater lung func- tion deterioration than patients with asthma without that mutation [3, 4]. However, several works published between 1998 and 2008 have been questioned since, on the basis that they report contradictory results [3–6]; furthermore, they had limitations that included incorrectly diagnosing asthma, using emergency registry databases, detecting just a single CFTR gene muta- tion and analysing different populations with unequal comparisons in terms of sex and age [5, 6]. Furthermore, all types of asthma were included, i.e., no phenotype filtering was applied. PLOS ONE | https://doi.org/10.1371/journal.pone.0251881 June 4, 2021 2 / 13 PLOS ONE CFTR gene, asthma and airway mucus hypersecretion CF is the most common severe autosomal recessive hereditary disease reported for Cauca- sians; in Spain incidence is estimated as 1 in 5,352 full-term births [7]. CF is caused by muta- tions in the CFTR gene residing in the long arm of human chromosome 7 and coding for the CFTR protein [8, 9], in turn, acting as a chloride channel on the surface of epithelial cells in a wide variety of organs (thereby explaining the wide range of clinical manifestations of CF). To date, of some 1,900 genetic variants described for the CFTR gene, most frequent in the Caucasian population is the three-base-pair deletion NM_000492.3(CFTR): c.1521_1523delCTT (p.Phe508del) or F508del according to the classical nomenclature [10, 11]. Since the inheritance pattern for CF is autosomal recessive, both copies of the mutated CFTR gene must be present for the disease to develop, whereas only a single copy is necessary to be a CF carrier. It is estimated that around 4%-5% of the general population are CF carriers [12]. The main line of work in our current project is the search for genetic variants (mutations or polymorphisms) in the CFTR gene using mass sequencing platforms and next-generation sequencing (NGS) and, more specifically, the investigation of airway mucus hypersecretors and non-hypersecretors with asthma and pre-defined clinical characteristics. The goal is, by considering the phenotype and genotype of the CFTR gene, to achieve better stratification in asthma classifications and so improve disease prognosis for hypersecreting patients and, ulti- mately, enhance the efficiency of current treatments and develop targeted and personalized future treatments. Materials and methods Comparative multicentre cross-sectional descriptive study, designed to determine the presence of genetic variants (mutations or polymorphisms) of the CFTR gene in hypersecreting and non-hypersecreting patients with asthma and to identify the clinical and inflammatory charac- teristics of hypersecreting patients. Included were 100 patients of both sexes, aged between 18 and 80 years, with and without airway mucus hypersecretion; all of them complied with asthma diagnostic criteria according to the Global Initiative for Asthma (GINA) guidelines published in 2009 [13] and none of them had a respiratory infection in the month before testing. Excluded were smokers and ex- smokers, patients with lung conditions (tuberculosis sequelae, bronchiectasis, CF, residual pleural diseases, interstitial diseases and severe associated comorbidities) and patients treated with oral corticosteroids or other immunomodulators for reasons other than asthma. Included patients were administered a questionnaire to evaluate symptoms and type of expectoration (S1 Appendix). Asthmatic hypersecretion was defined as the presence of cough productive of sputum on most days for at least 3 months in 2 successive years. Ethical considerations The study was conducted according to the principles of the Declaration of Helsinki (18th World Medical Assembly) and was approved by the Hospital de la Santa Creu i Sant Pau (HSCSP, Barcelona) Clinical Research Ethics Committee (approval number COD: HBSP-CFT-2014-68; ClinicalTrials.gov Identifier: NCT02558127). All patients signed an informed consent prior to participating in the study and were guaranteed the confidentiality of their data. Method Included between 2014 and 2016 were 100 patients with asthma (39 hypersecretors and 61 non-hypersecretors), 98 of whom were analysed (2 withdrew from the study). The patients PLOS ONE | https://doi.org/10.1371/journal.pone.0251881 June 4, 2021 3 / 13 PLOS ONE CFTR gene, asthma and airway mucus hypersecretion included in the study, who came from the outpatient clinics of the hospitals participating in the study, were recruited according to the criteria of the participating physicians. Exclusion criteria were the presence of other associated lung conditions (allergic bronchopulmonary aspergillosis, tuberculosis sequelae, severe bronchiectasis secondary to a respiratory disease other than asthma, CF, residual pleural diseases and interstitial diseases); severe associated comorbidities; and treatment with oral corticosteroids or other immunomodulators for causes other than asthma. Tests were performed as follows: fractional exhaled nitric oxide (FeNO), spirometry, induced sputum cell count (conducted only at the HSCSP), total immunoglobulin E (IgE), peripheral blood eosinophil count, C-reactive protein, blood fibrinogen, blood albumin and skin prick test for common pneumoallergens. All patients completed the validated Spanish version of the Asthma Control Test (ACT) questionnaire [14] and the Mini Asthma Quality of Life (Mini-AQLQ) questionnaire [15]. The sweat chloride test could not be performed, first because we do not have the necessary equipment for adults, and second, because when carry- ing out a mass sequencing genetic study, if the patient had CF, this would have been detected. FeNO was measured before spirometry using electrochemical equipment (NO Vario Ana- lyzer, FILT Lungen and Thorax Diagnostic GmbH, Berlin, Germany) on the basis of expira- tion yielding a continuous flow of 50 mL/s of total lung capacity in accordance with American Thoracic Society (ATS)/European Respiratory Society (ERS) recommendations from 2005 [16]. A high FeNO value was defined as �50 parts per billion (ppb) [17]. Spirometry was per- formed with a Datospir-600 spirometer (Sibelmed SA, Barcelona, Spain) by an experienced pulmonary function technician who applied Spanish Society of Pulmonology and Thoracic Surgery (SEPAR) recommendations [18, 19]. Induced sputum samples were collected, accord- ing to the ERS consensus protocol [20], using an ultrasonic nebulizer (Omron NE U07, HEALTHCARE Europe, Germany)–output 3 mL/s and mass mean aerodynamic diameter (MMAD) particle size 7 μm–to produce a hypertonic saline spray which the patient inhaled at increasing concentrations (3%, 4% and 5%). Sputum was processed within 2 hours of induc- tion. Patients were classified as having neutrophilic asthma when neutrophil count was �61%, eosinophilic asthma when eosinophil count was �3%, paucigranulocytic asthma when neutro- phil count was <61% and eosinophil count was <3%, and mixed asthma when neutrophil count was �61% and eosinophil count was �3% [21]. Total serum IgE was measured by ImmunoCAP using the UniCAP 250 system (Phadia AB, Uppsala, Sweden), with a high value defined as >160 IU/mL. The prick test, performed as standard, was considered positive for wheal diameters >3 mm [22]. Controlled asthma was defined as an ACT score �20. A high dose of inhaled glucocorticoids was defined as �1,000 mg/day of beclomethasone dipropio- nate or equivalent [23]. Study of genetic variants in the CFTR gene A 10-mL peripheral venous blood sample was extracted by the saline method from patients for analysis by the genetics service of the reference hospital. DNA was analysed for genetic variants of the CFTR gene by means of NGS using the Illumina platform MiSeq sequencer and pre- designed Multiplicom CFTR gene panels (CFTR Master Dx kit) as diagnostic tests, validated by a CE-IVD certificate and compatible with Illumina NGS sequencers. The NGS workflow consisted of 5 main steps: (1) library preparation using the CFTR Master Dx and MID Dx kits specific to the Illumina MiSeq for sample analyses; (2) cluster generation; (3) sequencing; (4) data analysis with the MiSeq Reporter software pre-installed in the MiSeq sequencer; and (5) data interpretation using computerized systems and databases incorporated in analytical soft- ware. Alignment and variant calling were implemented using standard methodologies. Briefly, PLOS ONE | https://doi.org/10.1371/journal.pone.0251881 June 4, 2021 4 / 13 PLOS ONE CFTR gene, asthma and airway mucus hypersecretion the reads were aligned against GRCh37 human genome assembly using Burrows-Wheeler Aligner (BWA) software. Variant calling was performed using the Genome Analysis Toolkit (GATK). Variant quality filtering parameters included a minimum of 30 reads, with at least 30% with the variant to call heterozygous genotypes. To prioritize variants, minor allele fre- quency (MAF) in the Genome Aggregation Database <0.05 was applied. Common variants were excluded by investigating the dbSNP, 1000G, ExAc and GnomAD v.2.1 databases. Mean vertical coverage was 1046X and mean horizontal coverage was 98.8% (Q�30). Steps 2–4 were performed automatically by the MiSeq sequencer. The genetic variants identified using NGS were validated by Sanger sequencing. In parallel, possible complete or partial CFTR gene deletions and/or duplications were identified using Multiplex Ligated Probe Amplification (MLPA). Statistical analysis Descriptive baseline values are reported as frequencies and percentages for qualitative data and means and standard deviation (SD) for quantitative data. The 2 asthma groups (hyperse- cretors and non-hypersecretors) were compared in terms of means and SD using the student-t test for the main variable and the remaining quantitative variables. The categorical variables, described by means of contingency tables and tested for differences using the chi-square test, are reported as numbers and percentages. Statistical significance was set to 5% (α = 0.05). Given the exploratory nature of the study, no correction was applied to multiple comparisons for over-dimensioning of the type I error. Statistical analyses were performed using SPSS (ver- sion 19.0) for Windows (SPSS, Inc., Chicago, Il, USA). Results Genetic results Of the 98 DNA samples obtained from the peripheral blood of patients with asthma, 97 were analysed and 1 was excluded as being unsuitable for processing. A total of 50 genetic variants were found in the CFTR gene in 86 patients (the remaining 12 patients did not show any of these genetic variants: 11 were non-hypersecretors and the remaining case had an unknown phenotype). Significant differences were only observed between the 2 groups in a single genetic variant, namely NM_000492.3 (CFTR): c.1680-870T>A, present in hypersecretors but not in non-hypersecretors: 78.94% vs. 59.32% in the majority allele (thymine nucleotide), and 21.05% vs. 40.67% in the minor allele (adenine nucleotide) (p = 0.036) (see Table 1). Several samples had more than a single variant of the CFTR gene (86 patients). Two patients, one of whom was patient #4 in Table 2, showed 2 changes in the CFTR gene; the other patient had 2 changes in heterozygosis (c.220C>T and c.3808G>A), both predicting amino acid changes–p.(Arg74Trp) and p.(Asp1270Asn)–in the protein encoded by the CFTR gene. The clinical effect associated with both genetic variants is defined as having varying clinical consequences– 15% and 17% pancreatic insufficiency, respectively–according to the CFTR2 database (2020). Of the 97 samples analysed, therefore, 4 showed pathological mutations (Table 2) already described in the literature and associated with CF. As all these mutations were detected in het- erozygosis, patients who were carriers of the disease were informed. This finding confirms the internal validity of our results, since the proportion coincides with the 4%-5% risk of being a CF carrier described in the literature for the Caucasian population [12]. As a secondary objective of the study, polymorphisms were analysed in relation to asthma severity, with significant differences observed for 2 polymorphisms, namely, NM_000492.3 (CFTR): c.2506G>T (p.Asp836Tyr) and NM_000492.4(CFTR): c.3140-92T>C (p = 0.024 and PLOS ONE | https://doi.org/10.1371/journal.pone.0251881 June 4, 2021 5 / 13 PLOS ONE CFTR gene, asthma and airway mucus hypersecretion Table 1. Main polymorphisms associated with the CFTR gene in patients with asthma with and without airway mucus hypersecretion. Genetic variant Hypersecretors Non-hypersecretors (N = 59) p (N = 38) c.1680-870T>A 21,05%/78,94% 40,67%/59,32% 0.036 (N = 8/30) (N = 24/35) c.1680-871A>G 100%/0% 98.3%/1.69% 0.608 (N = 38/0) (N = 58/1) c.1727G>C [p.(Gly576Ala)] 94.73%/5.26% 98.3%/1.69% 0.339 (N = 36/2) (N = 58/1) c.2002C>T [p.(Arg668Cys)] 92.10%/7.89% 98.3%/1.69% 0.165 (N = 35/3) (N = 58/1) c.2047_2052delAAAAAAinsAAAAG [p.(Lys684Serfs� 38)] 97.36%/2.63% 100%/ 0.392 (N = 37/1) (N = 59) c.2260G>A [p.(Val754Met)] 97.36%/2.63% 100%/ 0.392 (N = 37/1) (N = 59) c.2506G>T [p.(Asp836Tyr)] 97.36%/2.63% 94.91%/5.08% 0.488 (N = 37/1) (N = 56/3) c.2562T>G (p. =) 50%/50% 54.23%/45.76% 0.421 (N = 19/19) (N = 32/27) c.2619+3A>G 100%/ 98.3%/1.69% 0.608 (N = 38/0) (N = 58/1) c.2619+85_2619+86delAT 44.73%/55.26% 54.23%/45.76% 0.24 (N = 17/21) (N = 32/27) c.2619+106T>A 86.84%/13.55% 86.44%/13.55% 0.604 (N = 33/5) (N = 51/8) c.2909-71G>C 97.36%/2.63% 91.52%/8.47% 0.238 (N = 37/1) (N = 54/5) c.2909-92G>A 65.78%/34.21% 71.18%/28.81% 0.366 (N = 25/13) (N = 42/17) c.2991G>C [p.(Leu997Phe)] 100%/ 98.3%/1.69% 0.608 (N = 38) (N = 58/1) c.3139+42A>T 100%/ 98.3%/1.69% 0.608 (N = 38) (N = 58/1) c.3140-92T>C 97.36%/2.63% 89.83%/10.16% 0.16 (N = 37/1) (N = 53/6) c.3285A>T [p.(Thr1095Thr)] 97.36%/2.63% 100%/ 0.392 (N = 37/1) (N = 59) c.3367+37G>A 97.36%/2.63% 100%/ 0.392 (N = 37/1) (N = 59) c.3705T>G [p.(Ser1235Arg)] 92.10%/7.89% 96.61%/3.38% 0.299 (N = 35/3) (N = 57/2) c.3808G>A [p.(Asp1270Asn)] 100%/ 98.3%/1.69% 0.808 (N = 38) (N = 58/1) c.3870A>G 97.36%/2.63% 91.52%/8.47% 0.238 (N = 37/1) (N = 54/5) c.3874-200G>A 94.74%/5.26% 94.91%/5.08% 0.654 (N = 36/2) (N = 56/3) c.3909C>G [p.(Asn1303Lys)] 100%/ 98.3%/1.69% 0.608 (N = 38) (N = 58/1) (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0251881 June 4, 2021 6 / 13 PLOS ONE CFTR gene, asthma and airway mucus hypersecretion Table 1. (Continued) Genetic variant Hypersecretors Non-hypersecretors (N = 59) p (N = 38) c.4137-139G>A 57.89%/42.10% 71.18%/28.81% 0.13 (N = 22/16) (N = 42/17) c.4243-20A>G 100%/ 98.3%/1.69% 0.608 (N = 38) (N = 58/1) CFTR: cystic fibrosis transmembrane conductance regulator. https://doi.org/10.1371/journal.pone.0251881.t001 p = 0.049, respectively), more frequently present in majority alleles in patients with severe per- sistent asthma, and absent or present to a lesser extent in minority alleles (Table 3). Clinical hypersecretor and non-hypersecretor phenotypes The clinical, inflammatory and functional characteristics of hypersecretor and non-hyperse- cretor patients with asthma are shown in Table 4; of the 98 patients analysed, 39 were hyperse- cretors and 59 were non-hypersecretors. To a significant degree (p<0.05), hypersecretors compared to non-hypersecretors were older, had severer asthma, experienced greater bron- chial obstruction, had poorer asthma control (ACT <20), had received more short-term oral glucocorticoid treatments in the previous year, had lower peripheral blood albumin levels, induced sputum lymphocyte levels and IgE levels, and were less likely to have prick test-posi- tive asthma. Discussion Our study reflects that asthma associated with airway mucus hypersecretion may be linked to an intronic CFTR gene polymorphism (NM_000492.3 (CFTR): c.1680-870T>A). It should be noted, however, that the sample used for this study may not be representative, so this associa- tion, despite being significant, cannot be considered to be a definite linkage. Nonetheless, the patients with this mutation–older, with more severe asthma and with poorer clinical control– represented a non-allergic predominantly eosinophilic inflammatory phenotype. Airway mucus hypersecretion is a frequent symptom in patients with asthma, but unlike COPD, it is not usually taken into account in clinical practice or in clinical trials. Several pub- lished works have demonstrated that airway mucus hypersecretion is associated with greater asthma severity, is a marker of reduced lung function in both smokers and non-smokers, is associated with an increased number of exacerbation episodes and is a predictor of a poorer response to anti-inflammatory treatment with glucocorticoids [24–26]. Interestingly, in a Table 2. Pathological mutations described in the analysed population with asthma. Patient No. Mutation Exon 1 c.2047_2052delAAAAAAinsAAAAG [p.(Lys684Serfs� 38)] E14 2 c.3909C>G [p.(Asn1303Lys)] E24 3 c.1521_1523delCTT [p.(Phe508del)] E11 4 c.350G>A [p.(Arg117His)]; c.1521_1523delCTT [p.(Phe508del)] E4; E11 Patient #4 presented with 2 heterozygous changes: pathological (c.1521_1523delCTT) and change c.350G>A, the latter classified as missense since an amino acid change is predicted, although it is described in the cystic fibrosis database as a genetic variant with variable clinical consequences. The 4 patients with cystic fibrosis presented with a pathological mutation in heterozygosis. https://doi.org/10.1371/journal.pone.0251881.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0251881 June 4, 2021 7 / 13 PLOS ONE CFTR gene, asthma and airway mucus hypersecretion Table 3. Polymorphisms associated with the CFTR gene according to asthma severity. GENOTYPE Intermittent asthma Persistent asthma Mild Moderate Severe (N = 25) (N = 25) (N = 11) (N = 36) c.2506G>T [p.(Asp836Tyr)]� Homozygous majority allele (G/G) 23.70% 25.80% 11.80% 38.70% Minority allele present (G/T & T/T) 75% 25% 0% 0% c.3140-92T>C�� Homozygous majority allele (T/T) 23.30% 25.60% 12.20% 38.90% Minority allele present (T/C & C/C) 57.10% 28.60% 0% 14.30% CFTR: cystic fibrosis transmembrane conductance regulator. � p = 0.024 �� p = 0.049. https://doi.org/10.1371/journal.pone.0251881.t003 Table 4. Demographic, clinical and functional characteristics of asthma with and without airway mucus hypersecretion. Variables Asthma with hypersecretion Asthma without hypersecretion p (N = 39) (N = 59) Age (y) 57.43 (11.47) 49.44 (15.4) 0.004 Sex (% women) 61.5% 49.15% 0.159 Asthma diagnosis (% adults) 76.92% 71.18% 0.349 BMI (kg/m2) 27.41 (4.46) 27.24 (4.93) 0.864 Severe asthma (%) 58.97% 23.72% 0.005 FEV1/FVC (%) 64.39 (13.28) 69.55 (9.61) 0.041 FeNO (ppb) 32.45 (25.64) 39.81 (43.27) 0.291 Positive bronchodilator test (%) 23.68% 35.08% 0.483 Emergency visits, last 12 months 2.46 (3.08) 1.48 (2.24) 0.074 Oral glucocorticoid treatments, last 12 months 3.6 (3.7) 0.86 (1.3) 0.002 Medium-high oral glucocorticoid doses (%) 74.35% 64.4% 0.678 Rhinitis (%) 61.53% 64.44% 0.360 Polyposis (%) 20.55% 8.62% 0.172 High quality induced sputum (%) 61% 27.3% 0.100 Inflammatory phenotype paucigranulocytic: 31.25% paucigranulocytic: 26.32% 0.596 in induced sputum (%) neutrophilic: 12.5% neutrophilic: 26.32% eosinophilic: 56.25% eosinophilic: 47.36% (N = 16) (N = 19) Positive prick test (%) 46.15% 64.4% 0.216 Blood IgE (IU/mL) 126.4 (197) 407.59 (627.6) 0.003 Absolute eosinophils in peripheral blood (x10E9/L) 0.39 (0.32) 0.35 (0.27) 0.491 Blood polymerase chain reaction (mg/L) 4.26 (5.57) 4.20 (6.56) 0.969 Blood fibrinogen (g/L) 4.05 (1.02) 3.98 (0.93) 0.777 Blood albumin (g/L) 42.21 (3.06) 44.14 (3.12) 0.008 Lymphocytes in induced sputum (%) 0.71% (0.52) 1.05% (0.38) 0.023 ACT <20 (%) 58.3% 29.09% 0.021 AQLQ 3.55 (2.63) 2.6 (2.62) 0.113 Values are reported as means (standard deviation) or percentages, as indicated. ACT = Asthma Control Test; AQLQ = Asthma Quality of Life Questionnaire; BMI = Body mass index; FeNO = fractional exhaled nitric oxide; FEV1 = forced expiratory volume in the first second; FVC = forced vital capacity; IgE = immunoglobulin E. https://doi.org/10.1371/journal.pone.0251881.t004 PLOS ONE | https://doi.org/10.1371/journal.pone.0251881 June 4, 2021 8 / 13 PLOS ONE CFTR gene, asthma and airway mucus hypersecretion 10-year follow-up study of 13,756 patients with obstructive lung conditions (chronic bronchi- tis, emphysema and asthma), Lange et al. [27] observed that airway mucus hypersecretion increased the all-cause mortality risk and that hypersecretion combined with an altered lung function reflected a greater mortality risk for patients with asthma and COPD. Those results would point to the importance of airway mucus hypersecretion in influencing not only the natural course of an obstructive disease but also the associated mortality. While airway hypersecretion may be an underappreciated condition, clinical practice guidelines for asthma [13] highlight mucus along with bronchoconstriction and inflammation as causes of airway obstruction and airflow limitation. Possible explanations are related to (1) the role played by mucins, (2) the function of toll-like receptors, and (3) the fact of being a car- rier of a single-nucleotide polymorphism (SNP) as well as CFTR gene mutations. Our findings point to clinical characteristics that differentiated between patients with and without mucus hypersecretion, mainly that hypersecretors were older, had severer asthma, greater bronchial obstruction and poorer asthma control, had lower peripheral blood albumin and lower IgE levels, had lower induced sputum lymphocyte levels, were less likely to have prick test-positive asthma and, finally, needed more short-term oral glucocorticoid treatments in the previous year. Our results corroborate those of Martı́nez-Rivera et al. [28], who reported that hyperse- cretion, common in patients with asthma, was associated with greater airway obstruction, poorer asthma control and more exacerbation episodes. Another European prospective respi- ratory health study followed up over 9 years reported that a determinant of asthma severity was airway mucus hypersecretion [29]. Furthermore, Wheaterall et al. [1], who identified 5 phenotypes, observed that patients with phenotype 5 shared COPD and asthma characteristics (bronchial hyperresponsiveness, elevated FeNO, rhinitis, dermatitis and blood eosinophilia), airway mucus hypersecretion and a poorer response to glucocorticoid anti-inflammatory treatment (resulting in a greater need for treatment and more frequent hospital admissions due to exacerbation episodes). Chronic airway mucus hypersecretion is classically associated with bronchiectasis and with smoking, although only a minority of smokers develop this symptomatology. A plausible explanation for a greater propensity to hypersecretion may be genetic susceptibility. Dijkstra et al. [30] conducted a genome-wide association study of habitual or former smokers (�20 pack-years), with (n = 2,704) and without (n = 7,624) chronic mucus hypersecretion, report- ing, for all cohorts, a strong association between chronic mucus hypersecretion and SNP rs6577641 located in intron 9 of the special AT-rich sequence-binding protein 1 locus (SATB1) gene on chromosome 3 (p = 4.25×10−6; OR = 1.17). Likewise reported was that the risk allele was associated with greater mRNA expression of SATB1 in lung tissue (4.3×10−9) and that airway mucus hypersecretion was associated with greater mRNA expression of SATB1 in bronchial biopsies from patients with COPD [30]. Another genetic issue raised in the 1990s in relation to airway mucus hypersecretion and asthma was the correlation between carrying a CF mutation an increased asthma risk and a greater deterioration in lung function [3, 4]. Although the asthma-CF carrier relationship is well documented [3], studies carried out between 1998 and 2008 were widely debated, as some failed to find a higher incidence of asthma in CF carriers with the F508del mutation, although they did find a greater deteriora- tion in lung function in asthmatic CF carriers [5, 6]. It was also found that CFTR gene muta- tions altering RNA splicing and/or functional chloride conductance were likely to contribute to the susceptibility and pathogenesis of adult bronchiectasis and pulmonary non-tuberculous mycobacterial infection [31]. To date, of some 1,900 genetic variants described for the CFRT gene, that most frequently encountered in the Caucasian population is p.Phe508del (or F508del following the classical nomenclature). Worldwide only 20 mutations occur with a frequency greater than 0.1% [9, PLOS ONE | https://doi.org/10.1371/journal.pone.0251881 June 4, 2021 9 / 13 PLOS ONE CFTR gene, asthma and airway mucus hypersecretion 10]. Since the CF inheritance pattern is autosomal recessive, both copies of the mutated CFTR gene must be present for the disease to develop, whereas only a single copy is necessary to be a carrier. It is estimated that around 1 in 25–30 Caucasians are carriers of CF mutations–a higher frequency than for other ethnic and racial groups [7, 11, 32]. The fact that the NGS of 97 patients from our sample with asthma confirmed 4% to be CF carriers would tend to cor- roborate the internal validity of our genetic study. The use of NGS technology is a strength of this study–relative to studies that have typically sequenced just 39–50 CFTR gene mutations [3–6, 33–36]–as it permitted sequencing of the entire coding region of the CFTR gene along with flanking introns and regions for which pathogenic mutations have been described. Goodwin et al. [2] reported, for 4 cases of asthma and airway mucus hypersecretion, a neu- trophilic inflammatory phenotype accompanied by bronchiectasis, pansinusitis, respiratory infections and mutations and/or polymorphisms in the CFTR gene, positing the interesting possibility of an association between mutations and a characteristic phenotype. The results of our NGS study would suggest that, in hypersecretors compared to non-hypersecretors, the NM_000492.3 (CFTR): c.1680-870T>A polymorphism is present to a significantly greater degree: 78.94% vs. 59.32% in the majority allele (thymine nucleotide) and 21.05% vs. 40.67% in the minority allele (adenine nucleotide) (p = 0.036). This polymorphism is described as a SNP variant for CF [37], but given that its role in patients with asthma is still unknown, further stud- ies based on larger samples and specific clinical variables are needed to confirm our findings. The limitations of this study include the fact that the finding of a single polymorphism can potentially lead to spurious associations, among other reasons, because the variant might be found in linkage disequilibrium with 1 or more other variants and so constitute a characteristic haplotype. Information about haplotypes is becoming increasingly available online, so validat- ing the prognostic or therapeutic utility of this polymorphism would be useful. Other limita- tions are the lack of a questionnaire that objectively evaluated airway mucus hypersecretion, a possibly unrepresentative sample size and the fact that all the included patients were Caucasian (this may explain the multiple SNPs present in single patient). Strengths include–apart from the above-mentioned use of NGS for the DNA study, with the associated advantages–the inclu- sion of patients with a verified asthma diagnosis rather than patients from a database–as in previous studies–for whom a confirmed objective diagnosis may not have been available. A final strength is that exhaustive phenotyping was possible because patients underwent exten- sive clinical, functional and inflammatory testing. Conclusions The findings of this study of patients with asthma and airway mucus hypersecretion suggest the following: (1) a significantly high percentage of patients express the NM_000492.3(CFTR): c.1680-870T>A polymorphism in the CFTR gene, and (2) these patients tend to be older, have greater asthma severity and poorer clinical control and have a non-allergic inflammatory phe- notype. The findings point to a possibly interesting explanation: the overlap of having asthma and being a carrier of that CF gene mutation may cause a combination of asthma and an attenu- ated form of CF that would account for the component of bronchial mucosal hypersecretion. More studies are needed to validate the prognostic and therapeutic usefulness of these findings. Supporting information S1 Appendix. Questionnaires (Spanish and English) used to define bronchial hypersecre- tion in patients with asthma. (ZIP) PLOS ONE | https://doi.org/10.1371/journal.pone.0251881 June 4, 2021 10 / 13 PLOS ONE CFTR gene, asthma and airway mucus hypersecretion S2 Appendix. Statement regarding sample size representativeness and anonymous data. (ZIP) S3 Appendix. Tables. (ZIP) Acknowledgments The authors would like to thank Ailish M J Maher for translating and reviewing the article. Grateful thanks to the Emerging Asthma Group^, namely, as leaders, Vicente Plaza, Carolina Cisneros and José Serrano (emails: vplaza@santpau.cat; carol9199@yahoo.es; jserrano@separ.es) and as members, Astrid Crespo-Lessmann, Carlos Martı́nez- Rivera, Nuria Marina, Abel Pallarés-Sanmartı́n, Silvia Pascual, Juan Luis Garcı́a-Rivero, Alicia Padilla-Galo and Elena Curto. Author Contributions Conceptualization: Astrid Crespo-Lessmann, Carlos Martı́nez-Rivera, Alicia Padilla-Galo, Elena Curto, Carolina Cisneros, José Serrano, Vicente Plaza. Data curation: Carlos Martı́nez-Rivera, Nuria Marina, Abel Pallarés-Sanmartı́n, Silvia Pasc- ual, Juan Luis Garcı́a-Rivero, Alicia Padilla-Galo. Formal analysis: Astrid Crespo-Lessmann, Silvia Pascual. Funding acquisition: Astrid Crespo-Lessmann, José Serrano. Investigation: Sara Bernal, Ester Rojas, Nuria Marina, Abel Pallarés-Sanmartı́n, Silvia Pascual. Methodology: Sara Bernal, Elisabeth del Rı́o, Ester Rojas, Alicia Padilla-Galo. Project administration: Juan Luis Garcı́a-Rivero, Elena Curto, Carolina Cisneros, Montserrat Baiget, Vicente Plaza. Supervision: Carolina Cisneros, José Serrano, Vicente Plaza. Validation: Montserrat Baiget. Writing – original draft: Astrid Crespo-Lessmann. Writing – review & editing: Elena Curto, Vicente Plaza. References 1. Weatherall M, Travers J, Shirtcliffe PM, Marsh SE, Williams MV, Nowitz MR, et al. Distinct clinical phe- notypes of airways disease defined by cluster analysis. Eur Respir J 2009; 34: 812–8. https://doi.org/ 10.1183/09031936.00174408 PMID: 19357143 2. Goodwin Jodi, Spitale Naomi, Yaghi Asma, Dolovich Myrna, Nair Parameswaran. Cystic fibrosis trans- membrane conductance regulator gene abnormalities in patients with asthma and recurrent neutrophilic bronchitis. Can Respir J Vol 19 No 1 Jan/Feb 2012. https://doi.org/10.1155/2012/546702 PMID: 22332135 3. Morten Dahl A Tybjærg-Hansen, Lange Peter, Nordestgaard Børge G. 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