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We would like to express our gratitude to Chen and Yong1Chen Y.-J. Yong S.-B. Inhaled corticosteroid treatment's impact on asthma exacerbations: influence of human genetics on microbiome composition.J Allergy Clin Immunol. 2023; 152 (xxx): xxxGoogle Scholar for their interest in our article entitled “Human genetics influence microbiome composition involved in asthma exacerbations despite inhaled corticosteroid treatment”.2Perez-Garcia J. Espuela-Ortiz A. Hernández-Pérez J.M. González-Pérez R. Poza-Guedes P. Martin-Gonzalez E. et al.Human genetics influences microbiome composition involved in asthma exacerbations despite inhaled corticosteroid treatment.J Allergy Clin Immunol. 2023; 152: 799-806.e6Google Scholar We read with enthusiasm their suggestions to increase the robustness of the genetic associations with microbiome composition that we reported in individuals from the Genomics and Metagenomics of Asthma Severity (GEMAS) study.3Perez-Garcia J. Hernández-Pérez J.M. González-Pérez R. Sardón O. Martin-Gonzalez E. Espuela-Ortiz A. et al.The Genomics and Metagenomics of Asthma Severity (GEMAS) study: rationale and design.J Pers Med. 2020; 10: 123Google Scholar First, Chen and Yong1Chen Y.-J. Yong S.-B. Inhaled corticosteroid treatment's impact on asthma exacerbations: influence of human genetics on microbiome composition.J Allergy Clin Immunol. 2023; 152 (xxx): xxxGoogle Scholar recommended addressing the confounder effect of viral infections and vaccines, asthma traits and comorbidities, pharmacological treatment, and the role of certain bacterial species. We fully agree with the authors’ recommendations regarding the requirement to identify potential confounders in microbiome studies. Indeed, in our previous study in GEMAS,4Perez-Garcia J. González-Carracedo M. Espuela-Ortiz A. Hernández-Pérez J.M. González-Pérez R. Sardón-Prado O. et al.The upper-airway microbiome as a biomarker of asthma exacerbations despite inhaled corticosteroid treatment.J Allergy Clin Immunol. 2023; 151: 706-715Google Scholar we examined multiple clinical, demographic, and technical variables as microbiome confounders through univariate and multivariate permutational multivariate (PERMANOVA) tests on β-diversity distances. Some of the variables recommended by Chen and Yong1Chen Y.-J. Yong S.-B. Inhaled corticosteroid treatment's impact on asthma exacerbations: influence of human genetics on microbiome composition.J Allergy Clin Immunol. 2023; 152 (xxx): xxxGoogle Scholar were already examined and discarded as confounders during sample collection; these discarded variables include asthma comorbidities (ie, rhinitis, dermatitis, gastroesophageal reflux, and sleep apnea) and respiratory infections (ie, cold and flu).4Perez-Garcia J. González-Carracedo M. Espuela-Ortiz A. Hernández-Pérez J.M. González-Pérez R. Sardón-Prado O. et al.The upper-airway microbiome as a biomarker of asthma exacerbations despite inhaled corticosteroid treatment.J Allergy Clin Immunol. 2023; 151: 706-715Google Scholar We have now broadened the scope of the analyses to asthma severity, the presence of markers of type 2 inflammation,5Global strategy for asthma management and prevention. Global Initiative for Asthma.2022www.ginasthma.comDate accessed: July 25, 2023Google Scholar the history of pneumococcal and influenza vaccination in the past month, and the frequency of use of reliever medication in the past week. None of these covariates were identified as confounders for the bacterial communities (PMultivariate-PERMANOVA > .05, [Table I]). However, some limitations must be acknowledged. We were not able to examine the effect of asthma treatment in depth, given that information regarding the type of inhaled corticosteroid, dosage, or duration of therapy was not available for all of the individuals analyzed. Additionally, stratified analyses of asthma traits are restricted in the GEMAS study because more than 88% of patients have severe asthma with evidence of type 2 airway inflammation. Moreover, the role of bacterial species, their functionality, and their pathogenicity could not be investigated given the limitations of the 16S rRNA gene–targeted sequencing approach used.Table ISummary table of the assessment of multiple variables of interest as potential confounders of microbiome compositionVariableSalivaPharyngeal swabsNasal swabsUnivariate PERMANOVAMultivariate PERMANOVAUnivariate PERMANOVAMultivariate PERMANOVAUnivariate PERMANOVAMultivariate PERMANOVAPFr2P∗Significant P values are indicated in bold.PFr2P∗Significant P values are indicated in bold.PFr2P∗Significant P values are indicated in bold.Age.00113.8850.058.001.00112.8840.060.001.0013.1910.016.001Sex.0051.0190.004.348.0340.8570.004.566.0151.6720.008.049Antibiotics use.0014.6750.020.002.0014.3960.020.002.202NANANASequencing pool.0011.5610.020.019.0051.2530.012.155.0191.5970.016.026Season.0011.2030.015.171.0011.1340.016.221.0131.5310.023.013Body mass index.0091.0050.008.411.0070.9390.009.515.0031.5910.016.027Ever smoking.0011.5850.007.069.0011.6700.008.054.0071.4810.007.067Rhinitis.0051.6990.007.056.0011.5500.007.061.454NANANADermatitis.605NANANA.075NANANA.643NANANAGastroesophageal reflux.116NANANA.102NANANA.0261.2840.006.122Sleep apnea.0111.0180.004.395.0060.7010.003.842.249NANANAPre-FEV1 (%).0011.4810.006.109.0021.3860.006.127.032.7990.004.778Pre-FVC (%).0010.8280.003.630.0010.9770.005.463.0280.9780.005.433Cold.095NANANA.123NANANA.867NANANACavities.0112.2680.009.016.0032.1080.010.017.078NANANAMouth ulcers.871NANANA.839NANANA.164NANANATreatment with ICS†Variables recorded in the previous 30 minutes of biologic sample collection..088NANANA.223NANANA.0320.9880.005.421Water intake†Variables recorded in the previous 30 minutes of biologic sample collection..057NANANA.296NANANA.351NANANALiquid intake†Variables recorded in the previous 30 minutes of biologic sample collection..0291.0800.005.314.0122.6430.012.002.447NANANAEat food†Variables recorded in the previous 30 minutes of biologic sample collection..897NANANA.646NANANA.168NANANATeeth brush†Variables recorded in the previous 30 minutes of biologic sample collection..148NANANA.925NANANA.781NANANASmoke†Variables recorded in the previous 30 minutes of biologic sample collection..119NANANA.0071.7800.008.054.0102.1350.010.009Chewing gum†Variables recorded in the previous 30 minutes of biologic sample collection..0242.4630.010.013.934NANANA.120NANANAHousehold pets.0201.4840.006.104.343NANANA.108NANANAInfluenza vaccine.0011.0060.004.392.0011.0740.005.327.410NANANAPneumococcal vaccine.0181.3550.006.144.052NANANA.293NANANAT2-inflammation markers‡The presence of type 2 airway inflammation was defined as meeting 1 of the following criteria according to the Global Initiative for Asthma 2022 guideline adapted to available data in the GEMAS study: (1) a blood eosinophil count of at least 300 cells/μL, (2) a fractional exhaled nitric oxide level of at least 20 ppb, and (3) the presence of atopy. No evidence of type 2 airway inflammation was declared if none of these 3 criteria was met (or at least 2 of them in case of missing data)..067NANANA.0111.2850.006.181.231NANANAAsthma severity.056NANANA.260NANANA.428NANANASABA frequency use§Use of reliever medication in the past week for to asthma symptoms as reported in the asthma control questionnaire..120NANANA.253NANANA.0111.1290.006.249This table has been updated from Perez-Garcia et al4Perez-Garcia J. González-Carracedo M. Espuela-Ortiz A. Hernández-Pérez J.M. González-Pérez R. Sardón-Prado O. et al.The upper-airway microbiome as a biomarker of asthma exacerbations despite inhaled corticosteroid treatment.J Allergy Clin Immunol. 2023; 151: 706-715Google Scholar and includes 5 new covariates analyzed in this reply letter. A full description of the assessment of clinical variables has been described in Perez-Garcia et al.3Perez-Garcia J. Hernández-Pérez J.M. González-Pérez R. Sardón O. Martin-Gonzalez E. Espuela-Ortiz A. et al.The Genomics and Metagenomics of Asthma Severity (GEMAS) study: rationale and design.J Pers Med. 2020; 10: 123Google ScholarFVC, Forced vital capacity; ICS, Inhaled corticosteroid; NA, not applicable; SABA, short-acting β-agonist; T2, type 2.∗ Significant P values are indicated in bold.† Variables recorded in the previous 30 minutes of biologic sample collection.‡ The presence of type 2 airway inflammation was defined as meeting 1 of the following criteria according to the Global Initiative for Asthma 2022 guideline adapted to available data in the GEMAS study: (1) a blood eosinophil count of at least 300 cells/μL, (2) a fractional exhaled nitric oxide level of at least 20 ppb, and (3) the presence of atopy. No evidence of type 2 airway inflammation was declared if none of these 3 criteria was met (or at least 2 of them in case of missing data).§ Use of reliever medication in the past week for to asthma symptoms as reported in the asthma control questionnaire. Open table in a new tab This table has been updated from Perez-Garcia et al4Perez-Garcia J. González-Carracedo M. Espuela-Ortiz A. Hernández-Pérez J.M. González-Pérez R. Sardón-Prado O. et al.The upper-airway microbiome as a biomarker of asthma exacerbations despite inhaled corticosteroid treatment.J Allergy Clin Immunol. 2023; 151: 706-715Google Scholar and includes 5 new covariates analyzed in this reply letter. A full description of the assessment of clinical variables has been described in Perez-Garcia et al.3Perez-Garcia J. Hernández-Pérez J.M. González-Pérez R. Sardón O. Martin-Gonzalez E. Espuela-Ortiz A. et al.The Genomics and Metagenomics of Asthma Severity (GEMAS) study: rationale and design.J Pers Med. 2020; 10: 123Google Scholar FVC, Forced vital capacity; ICS, Inhaled corticosteroid; NA, not applicable; SABA, short-acting β-agonist; T2, type 2. Second, Chen and Yong1Chen Y.-J. Yong S.-B. Inhaled corticosteroid treatment's impact on asthma exacerbations: influence of human genetics on microbiome composition.J Allergy Clin Immunol. 2023; 152 (xxx): xxxGoogle Scholar requested further details on the genetic associations of microbiome traits in African Americans and Latinos from the replication phase. On one hand, all summary statistics of suggestive associations (P < 1 × 10–5), the annotated genes, and a full description of all significant enrichment terms evaluated in the replication phase are reported in the additional Tables 6, 7, and 8, which are publicly available in the Zenodo Repository (under accession no. 7472532). As in the discovery phase, no genetic variant surpassed the genome-wide significance threshold (P < 5 × 10–8) in African American and Latino individuals, being enrichment analyses an alternative approach to gain biologic insights into suggestive associations. On the other hand, we agree with Chen and Yong1Chen Y.-J. Yong S.-B. Inhaled corticosteroid treatment's impact on asthma exacerbations: influence of human genetics on microbiome composition.J Allergy Clin Immunol. 2023; 152 (xxx): xxxGoogle Scholar that our discovery and replication populations have distinctive demographic and clinical characteristics with a potential impact on microbiome composition. However, our findings support the view that genetic signatures underlying microbiome composition and asthma-related traits might be shared across diverse populations despite large differences in age and genetic ancestry. Finally, we agree with Chen and Yong1Chen Y.-J. Yong S.-B. Inhaled corticosteroid treatment's impact on asthma exacerbations: influence of human genetics on microbiome composition.J Allergy Clin Immunol. 2023; 152 (xxx): xxxGoogle Scholar that future microbiome studies should address the potential effect of coronavirus disease 2019 (COVID-19) infection and vaccine in bacterial communities. However, this would not apply to our study because all individuals from the GEMAS study and the replication populations were enrolled between March 2018 and March 2020 and between 2006 and 2014, respectively (ie, before the beginning of the global COVID-19 pandemic).2Perez-Garcia J. Espuela-Ortiz A. Hernández-Pérez J.M. González-Pérez R. Poza-Guedes P. Martin-Gonzalez E. et al.Human genetics influences microbiome composition involved in asthma exacerbations despite inhaled corticosteroid treatment.J Allergy Clin Immunol. 2023; 152: 799-806.e6Google Scholar In conclusion, we agree with Chen and Yong1Chen Y.-J. Yong S.-B. Inhaled corticosteroid treatment's impact on asthma exacerbations: influence of human genetics on microbiome composition.J Allergy Clin Immunol. 2023; 152 (xxx): xxxGoogle Scholar that confounder correction is essential to obtain reliable findings in microbiome studies. Our analyses demonstrated that most of the variables highlighted by Chen and Yong1Chen Y.-J. Yong S.-B. Inhaled corticosteroid treatment's impact on asthma exacerbations: influence of human genetics on microbiome composition.J Allergy Clin Immunol. 2023; 152 (xxx): xxxGoogle Scholar are not confounders of the microbiome composition in our study, although we recognized some limitations inherent to amplicon-targeted sequencing and variable recording. We also agree with with Chen and Yong1Chen Y.-J. Yong S.-B. Inhaled corticosteroid treatment's impact on asthma exacerbations: influence of human genetics on microbiome composition.J Allergy Clin Immunol. 2023; 152 (xxx): xxxGoogle Scholar regarding the need for more homogenous populations, and we support the need to generate more cohorts with the required data to gain a deeper understanding of the influence of genetic variation on the microbiome in patients with asthma. Supported by a grant (SAF2017-83417R) to M.P.-Y. and F.L.-D. funded by the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033) and “ERDF a Way of Making Europe”. M.P.-Y. was also funded by Consorcio Centro de Investigación Biomédica en Red, Instituto de Salud Carlos III, and the European Regional Development Fund (grant CB06/06/1088), and through the Ramón y Cajal Program (grant RYC-2015-17205) funded by the MCIN/AEI/10.13039/501100011033 and the European Social Fund “ESF Investing in Your Future”. J.P.-G. was supported by a fellowship (FPU19/02175 [Formación de Profesorado Universitario Program]) from the Spanish Ministry of Universities. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest. Inhaled corticosteroid treatment's impact on asthma exacerbations: Influence of human genetics on microbiome compositionJournal of Allergy and Clinical ImmunologyPreviewWe read with great interest the study by Perez-Garcia et al1 on the human genetics that influence microbiome composition involved in asthma exacerbations despite inhaled corticosteroid (ICS) treatment. In this retrospective cohort study using data from the Genomic and Metagenomics of Asthma Severity (GEMAS) study, Perez-Garcia et al1 hypothesized a relationship between the genetic variants, microbiome associated with asthma exacerbation, and possible biologic pathway. To augment their findings, we would like to offer several suggestions on covariates that could be adopted and included in their study. Full-Text PDF