Development and validation of exhaled breath condensate microRNAs to identify and endotype asthma in children

Autoři: Francisca Castro Mendes aff001;  Inês Paciência aff001;  António Carlos Ferreira aff004;  Carla Martins aff008;  João Cavaleiro Rufo aff001;  Diana Silva aff001;  Pedro Cunha aff009;  Mariana Farraia aff002;  Pedro Moreira aff002;  Luís Delgado aff001;  Miguel Luz Soares aff004;  André Moreira aff001
Působiště autorů: Serviço de Imunologia Básica e Clínica, Departamento de Patologia, Faculdade de Medicina da Universidade do Porto, Porto, Portugal aff001;  EPIUnit—Instituto de Saúde Pública, Universidade do Porto, Porto, Portugal aff002;  Institute of Science and Innovation in Mechanical Engineering and Industrial Management (INEGI), Porto, Portugal aff003;  Laboratório de Apoio à Investigação em Medicina Molecular (LAIMM), Faculdade de Medicina da Universidade do Porto, Porto, Portugal aff004;  Departamento de Biomedicina-Unidade de Biologia Experimental, Centro de Investigação Médica (CIM), Faculdade de Medicina da Universidade do Porto, Porto, Portugal aff005;  Pain Group, Instituto de Biologia Molecular e Celular (IBMC), Porto, Portugal aff006;  Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal aff007;  Serviço de Imunoalergologia, Centro Hospitalar São João, Porto, Portugal aff008;  Faculdade de Ciências da Nutrição e Alimentação da Universidade do Porto, Porto, Portugal aff009
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224983


Detection and quantification of microRNAs (miRNAs) in exhaled breath condensate (EBC) has been poorly explored. Therefore we aimed to assess miRNAs in EBC as potential biomarkers to diagnose and endotype asthma in school aged children. In a cross sectional, nested case control study, all the asthmatic children (n = 71) and a random sample of controls (n = 115), aged 7 to 12 years, attending 71 classrooms from 20 local schools were selected and arbitrarily allocated to the development or validation set. Participants underwent skin-prick testing, spirometry with bronchodilation, had exhaled level of nitric oxide determined and EBC collected. Based on previous studies eleven miRNAs were chosen and analyzed in EBC by reverse transcription-quantitative real-time PCR. Principal component analysis was applied to identify miRNAs profiles and associations were estimated using regression models. In the development set (n = 89) two clusters of miRNAs were identified. After adjustments, cluster 1 and three of its clustered miRNAs, miR-126-3p, miR-133a-3p and miR-145-5p were positively associated with asthma. Moreover miR-21-5p was negatively associated with symptomatic asthma and positively associated with positive bronchodilation without symptoms. An association was also found between miR-126-3p, cluster 2 and one of its clustered miRNA, miR-146-5p, with higher FEF25-75 reversibility. These findings were confirmed in the validation set (n = 97) where two identical clusters of miRNAs were identified. Additional significant associations were observed between miR-155-5p with symptomatic asthma, negative bronchodilation with symptoms and positive bronchodilation without symptoms. We showed that microRNAs can be measured in EBC of children and may be used as potential biomarkers of asthma, assisting asthma endotype establishment.

Klíčová slova:

Asthma – Biomarkers – Diagnostic medicine – MicroRNAs – Pediatrics – Phenotypes – Pulmonary function – RNA extraction


1. Sastre B, Cañas JA, Rodrigo-Muñoz JM, del Pozo V. Novel modulators of asthma and allergy: exosomes and microRNAs. Frontiers in Immunology. 2017;8(826).

2. Cruickshank-Quinn C, Armstrong M, Powell R, Gomez J, Elie M, Reisdorph N. Determining the presence of asthma-related molecules and salivary contamination in exhaled breath condensate. Resp Res. 2017;18(1):57.

3. Takaku Y, Kurashima K, Kobayashi T, Nakagome K, Nagata M. Eicosanoids in exhaled breath condensate of airway inflammation in patients with asthma. Allergol Int. 2016;65:S65–6. doi: 10.1016/j.alit.2016.05.007 27321647

4. Peel AM, Crossman-Barnes CJ, Tang J, Fowler SJ, Davies GA, Wilson AM, et al. Biomarkers in adult asthma: a systematic review of 8-isoprostane in exhaled breath condensate. J Breath Res. 2017;11(1):016011. doi: 10.1088/1752-7163/aa5a8a 28102831

5. Maes T, Cobos FA, Schleich F, Sorbello V, Henket M, De Preter K, et al. Asthma inflammatory phenotypes show differential microRNA expression in sputum. The Journal of allergy and clinical immunology. 2016;137(5):1433–46. doi: 10.1016/j.jaci.2016.02.018 27155035

6. Rebane A, Akdis CA. MicroRNAs: Essential players in the regulation of inflammation. The Journal of allergy and clinical immunology. 2013;132(1):15–26. doi: 10.1016/j.jaci.2013.04.011 23726263

7. Lacedonia D, Palladino GP, Foschino-Barbaro MP, Scioscia G, Carpagnano GE. Expression profiling of miRNA-145 and miRNA-338 in serum and sputum of patients with COPD, asthma, and asthma–COPD overlap syndrome phenotype. Int J Chron Obstruct Pulmon Dis. 2017;12:1811–7. doi: 10.2147/COPD.S130616 28694694

8. Lu TX, Rothenberg ME. MicroRNA. The Journal of allergy and clinical immunology. 2017;141(4):1202–7. doi: 10.1016/j.jaci.2017.08.034 29074454

9. Levanen B, Bhakta NR, Torregrosa Paredes P, Barbeau R, Hiltbrunner S, Pollack JL, et al. Altered microRNA profiles in bronchoalveolar lavage fluid exosomes in asthmatic patients. The Journal of allergy and clinical immunology. 2013;131(3):894–903. doi: 10.1016/j.jaci.2012.11.039 23333113

10. Panganiban RP, Wang Y, Howrylak J, Chinchilli VM, Craig TJ, August A, et al. Circulating microRNAs as biomarkers in patients with allergic rhinitis and asthma. The Journal of allergy and clinical immunology. 2016;137(5):1423–32. doi: 10.1016/j.jaci.2016.01.029 27025347

11. Cavaleiro Rufo J, Paciencia I, Silva D, Martins C, Madureira J, Oliveira Fernandes E, et al. Swimming pool exposure is associated with autonomic changes and increased airway reactivity to a beta-2 agonist in school aged children: A cross-sectional survey. PLoS One. 2018;13(3):e0193848. doi: 10.1371/journal.pone.0193848 29529048

12. Silva D, Severo M, Paciência I, Rufo J, Martins C, Moreira P, et al. Setting definitions of childhood asthma in epidemiologic studies. Pediatric Allergy and Immunology. 2019;0(ja).

13. Spycher BD, Silverman M, Kuehni CE. Phenotypes of childhood asthma: are they real? Clin Exp Allergy. 2010;40(8):1130–41. doi: 10.1111/j.1365-2222.2010.03541.x 20545704

14. Asher MI, Keil U, Anderson HR, Beasley R, Crane J, Martinez F, et al. International Study of Asthma and Allergies in Childhood (ISAAC): rationale and methods. Eur Respir J. 1995;8(3):483–91. doi: 10.1183/09031936.95.08030483 7789502

15. Kuczmarski RJ, Ogden CL, Grummer-Strawn LM, Flegal KM, Guo SS, Wei R, et al. CDC growth charts: United States. Adv Data. 2000(314):1–27. 11183293

16. Dweik RA, Boggs PB, Erzurum SC, Irvin CG, Leigh MW, Lundberg JO, et al. An official ATS clinical practice guideline: interpretation of exhaled nitric oxide levels (FENO) for clinical applications. Am J Respir Crit Care Med. 2011;184(5):602–15. doi: 10.1164/rccm.9120-11ST 21885636

17. Montuschi P. Analysis of exhaled breath condensate in respiratory medicine: methodological aspects and potential clinical applications. Ther Adv Respir Dis. 2007;1(1):5–23. doi: 10.1177/1753465807082373 19124344

18. Liu J, Thomas PS. Relationship between exhaled breath condensate volume and measurements of lung volumes. Respiration. 2007;74(2):142–5. doi: 10.1159/000094238 16804290

19. Yang X, Chen Q, Zeng J, Zhang J, Shaw C. A mass transfer model for simulating volatile organic compound emissions from ‘wet’coating materials applied to absorptive substrates. International journal of heat and mass transfer. 2001;44(9):1803–15.

20. Liu F, Qin HB, Xu B, Zhou H, Zhao DY. Profiling of miRNAs in pediatric asthma: upregulation of miRNA-221 and miRNA-485-3p. Mol Med Rep. 2012;6(5):1178–82. doi: 10.3892/mmr.2012.1030 22895815

21. Malmhall C, Johansson K, Winkler C, Alawieh S, Ekerljung L, Radinger M. Altered miR-155 Expression in Allergic Asthmatic Airways. Scand J Immunol. 2017;85(4):300–7. doi: 10.1111/sji.12535 28199728

22. Pinkerton M, Chinchilli V, Banta E, Craig T, August A, Bascom R, et al. Differential expression of microRNAs in exhaled breath condensates of patients with asthma, patients with chronic obstructive pulmonary disease, and healthy adults. The Journal of allergy and clinical immunology. 2013;132(1):217–9. doi: 10.1016/j.jaci.2013.03.006 23628339

23. Qin HB, Xu B, Mei JJ, Li D, Liu JJ, Zhao DY, et al. Inhibition of miRNA-221 suppresses the airway inflammation in asthma. Inflammation. 2012;35(4):1595–9. doi: 10.1007/s10753-012-9474-1 22572970

24. Suojalehto H, Lindstrom I, Majuri ML, Mitts C, Karjalainen J, Wolff H, et al. Altered microRNA expression of nasal mucosa in long-term asthma and allergic rhinitis. Int Arch Allergy Immunol. 2014;163(3):168–78. doi: 10.1159/000358486 24513959

25. Trinh HKT, Pham DL, Kim SC, Kim RY, Park HS, Kim SH. Association of the miR-196a2, miR-146a, and miR-499 Polymorphisms with Asthma Phenotypes in a Korean Population. Mol Diagn Ther. 2017;21(5):547–54. doi: 10.1007/s40291-017-0280-1 28527151

26. Wu XB, Wang MY, Zhu HY, Tang SQ, You YD, Xie YQ. Overexpression of microRNA-21 and microRNA-126 in the patients of bronchial asthma. Int J Clin Exp Med. 2014;7(5):1307–12. 24995087

27. Sinha A, Yadav AK, Chakraborty S, Kabra SK, Lodha R, Kumar M, et al. Exosome-enclosed microRNAs in exhaled breath hold potential for biomarker discovery in patients with pulmonary diseases. Journal of Allergy and Clinical Immunology. 2013;132(1):219–22.e7. doi: 10.1016/j.jaci.2013.03.035 23683467

28. Rijavec M, Korosec P, Zavbi M, Kern I, Malovrh MM. Let-7a is differentially expressed in bronchial biopsies of patients with severe asthma. Sci Rep. 2014;4:6103. doi: 10.1038/srep06103 25130484

29. Panganiban RP, Pinkerton MH, Maru SY, Jefferson SJ, Roff AN, Ishmael FT. Differential microRNA epression in asthma and the role of miR-1248 in regulation of IL-5. Am J Clin Exp Immunol. 2012;1(2):154–65. 23885321

30. Solberg OD, Ostrin EJ, Love MI, Peng JC, Bhakta NR, Hou L, et al. Airway epithelial miRNA expression is altered in asthma. Am J Respir Crit Care Med. 2012;186(10):965–74. doi: 10.1164/rccm.201201-0027OC 22955319

31. Williams AE, Larner-Svensson H, Perry MM, Campbell GA, Herrick SE, Adcock IM, et al. MicroRNA expression profiling in mild asthmatic human airways and effect of corticosteroid therapy. PLoS One. 2009;4(6):e5889. doi: 10.1371/journal.pone.0005889 19521514

32. Papi M, Caracciolo GJNT. Principal component analysis of personalized biomolecular corona data for early disease detection. 2018;21:14–7.

33. Maitra S, Yan JJAMSM. Principle component analysis and partial least squares: Two dimension reduction techniques for regression. 2008;79:79–90.

34. McGeachie MJ, Davis JS, Kho AT, Dahlin A, Sordillo JE, Sun M, et al. Asthma remission: Predicting future airways responsiveness using an miRNA network. The Journal of allergy and clinical immunology. 2017;140(2):598–600.e8. doi: 10.1016/j.jaci.2017.01.023 28238746

35. Forno E, Celedon JC. Epigenomics and Transcriptomics in the Prediction and Diagnosis of Childhood Asthma: Are We There Yet? Front Pediatr. 2019;7:115. doi: 10.3389/fped.2019.00115 31001502

36. Lødrup Carlsen KC, Pijnenburg M. Identification of asthma phenotypes in children. 2011;8(1):38–44.

37. Loureiro CC, Sa-Couto P, Todo-Bom A, Bousquet J. Cluster analysis in phenotyping a Portuguese population. Rev Port Pneumol (2006). 2015.

38. Depner M, Fuchs O, Genuneit J, Karvonen AM, Hyvarinen A, Kaulek V, et al. Clinical and epidemiologic phenotypes of childhood asthma. Am J Respir Crit Care Med. 2014;189(2):129–38. doi: 10.1164/rccm.201307-1198OC 24283801

39. Pavord ID, Beasley R, Agusti A, Anderson GP, Bel E, Brusselle G, et al. After asthma: redefining airways diseases. Lancet. 2018;391(10118):350–400. doi: 10.1016/S0140-6736(17)30879-6 28911920

40. Milger K, Gotschke J, Krause L, Nathan P, Alessandrini F, Tufman A, et al. Identification of a plasma miRNA biomarker signature for allergic asthma: A translational approach. Allergy. 2017;72(12):1962–71. doi: 10.1111/all.13205 28513859

41. Rodrigo-Munoz JM, Canas JA, Sastre B, Rego N, Greif G, Rial M, et al. Asthma diagnosis using integrated analysis of eosinophil microRNAs. Allergy. 2018;74(3):507–17. doi: 10.1111/all.13570 30040124

42. Prats-Puig A, Ortega FJ, Mercader JM, Moreno-Navarrete JM, Moreno M, Bonet N, et al. Changes in circulating microRNAs are associated with childhood obesity. J Clin Endocrinol Metab. 2013;98(10):E1655–60. doi: 10.1210/jc.2013-1496 23928666

43. Ong J, Woldhuis RR, Boudewijn IM, van den Berg A, Kluiver J, Kok K, et al. Age-related gene and miRNA expression changes in airways of healthy individuals. Sci Rep. 2019;9(1):3765. doi: 10.1038/s41598-019-39873-0 30842487

44. Karam RA, Abd Elrahman DM. Differential expression of miR-155 and Let-7a in the plasma of childhood asthma: Potential biomarkers for diagnosis and severity. Clin Biochem. 2019.

45. Heffler E, Allegra A, Pioggia G, Picardi G, Musolino C, Gangemi S. MicroRNA Profiling in Asthma: Potential Biomarkers and Therapeutic Targets. Am J Respir Cell Mol Biol. 2017;57(6):642–50. doi: 10.1165/rcmb.2016-0231TR 28489455

46. Jardim MJ, Dailey L, Silbajoris R, Diaz-Sanchez D. Distinct microRNA expression in human airway cells of asthmatic donors identifies a novel asthma-associated gene. Am J Respir Cell Mol Biol. 2012;47(4):536–42. doi: 10.1165/rcmb.2011-0160OC 22679274

47. Hammad Mahmoud Hammad R, Hamed D, Eldosoky M, Ahmad A, Osman HM, Abd Elgalil HM, et al. Plasma microRNA-21, microRNA-146a and IL-13 expression in asthmatic children. Innate Immun. 2018;24(3):171–9. doi: 10.1177/1753425918763521 29635981

48. Zhou H, Li J, Gao P, Wang Q, Zhang J. miR-155: A Novel Target in Allergic Asthma. International journal of molecular sciences. 2016;17(10):1773.

49. Mattes J, Collison A, Plank M, Phipps S, Foster PSJPotNAoS. Antagonism of microRNA-126 suppresses the effector function of TH2 cells and the development of allergic airways disease. 2009;106(44):18704–9.

50. Fan L, Wang X, Fan L, Chen Q, Zhang H, Pan H, et al. MicroRNA-145 influences the balance of Th1/Th2 via regulating RUNX3 in asthma patients. 2016;42(8–10):417–24. doi: 10.1080/01902148.2016.1256452 27902892

51. Charlotte N, James SH, GM P. A guide to miRNAs in inflammation and innate immune responses. The FEBS Journal. 2018;285(20):3695–716. doi: 10.1111/febs.14482 29688631

52. Cavaleiro Rufo J, Paciencia I, Mendes FC, Farraia M, Rodolfo A, Silva D, et al. Exhaled breath condensate volatilome allows sensitive diagnosis of persistent asthma. Allergy. 2019;74(3):527–34. doi: 10.1111/all.13596 30156012

53. Gahleitner F, Guallar-Hoyas C, Beardsmore CS, Pandya HC, Thomas CP. Metabolomics pilot study to identify volatile organic compound markers of childhood asthma in exhaled breath. Bioanalysis. 2013;5(18):2239–47. doi: 10.4155/bio.13.184 24053239

54. Carraro S, Giordano G, Reniero F, Carpi D, Stocchero M, Sterk PJ, et al. Asthma severity in childhood and metabolomic profiling of breath condensate. Allergy. 2013;68(1):110–7. doi: 10.1111/all.12063 23157191

55. Maniscalco M, Paris D, Melck DJ, D'Amato M, Zedda A, Sofia M, et al. Coexistence of obesity and asthma determines a distinct respiratory metabolic phenotype. Journal of Allergy and Clinical Immunology. 2017;139(5):1536–47.e5. doi: 10.1016/j.jaci.2016.08.038 27746236

Článek vyšel v časopise


2019 Číslo 11