BACKGROUND: Despite the construction of the metagenome of the asthmatic lung, limitations persist in sampling the bronchial airway. This study analyzed extracellular vesicles (EVs) obtained from exhaled breath condensate (EBC) to compare the distinct characteristics of the microbiome in asthmatics with those in healthy controls and proposed a diagnostic artificial intelligence-based model of asthma. METHODS: We obtained the EBC from 58 healthy controls and 251 patients with asthma. EVs were isolated from the EBC and analyzed. The extracted 16s rDNA was subjected to next generation sequencing. Taxonomic profiling was conducted for all samples at the genus level. A combination of artificial neural network (ANN) and gradient boosting (GBM) was applied to selective EBC biomarkers. RESULTS: The asthma group exhibited significantly higher alpha diversity based on the results of the Chao1, Shannon, and Simpson indices. The bacterial composition of patients with asthma different from that of the controls. At the genus level, Sphingomonas, Akkermansia, Methylophaga, Acidocella, and Marinobacter were significantly more abundant in patients with asthma. The diagnostic model using GBM and ANN demonstrated good performance with respective areas under the curve of 0.832 and 0.769. Firmicutes and Proteobacteria at the phylum level were common important features between the GBM and ANN asthma models. CONCLUSION: We demonstrated a distinct pattern in the microbiome of patients with asthma, indicating the potential role of microbiome-based diagnosis of asthma. To the best of our knowledge, this was the first study to identify the microbiome in asthma using EBC-derived EVs.

Serum MRGPRX2 as a long-time available biomarker for drug-induced anaphylaxisTo the Editor,The diagnosis of anaphylaxis is based on clinical symptoms after exposure to a trigger factor1. Serum tryptase is useful for an accurate diagnosis of anaphylaxis. However, one of its limitations is short half-life, hence the need for blood samples to be obtained at the onset of anaphylaxis2,3. Mas-related G protein-coupled receptor-X2 (MRGPRX2), expressed in mast cells, is related to degranulation of mast cells in the IgE-independent pathway for anaphylaxis4-6. We aimed to evaluate serum MRGPRX2 levels in patients with drug-induced anaphylaxis (DIA) and to determine usefulness as a potential marker of anaphylaxis by maintaining its concentrations longer.We included 68 patients who consulted at allergy clinic for a history of anaphylaxis after causal drug administration between January 2010 and December 2017. Diagnosis of DIA and causal drugs were determined by an allergist. Patients were excluded if 1) causal drugs were ambiguous, or 2) other relevant causes were combined. Each patient underwent serum MRGPRX2 level measurement randomly at least once from the onset of anaphylaxis. MRGPRX2 levels were determined using an ELISA kit from MyBioSource. We also measured MRGPRX2 levels of 74 controls.We classified DIA patients according to causal drugs to investigate the difference in MRGPRX2 levels between drugs. In addition, we analyzed the MRGPRX2 levels according to the time interval from the anaphylactic event to verify maintenance of MRGPRX2 levels over a longer period of time. The time interval for each patient was defined as the gap between the measurement time of MRGPRX2 levels and the anaphylactic event. This study was approved by the institutional review board of Asan Medical Center (2018-1252), and subjects provided written informed consent.This study included 74 controls and 68 patients with DIA. The mean MRGPRX2 level in the DIA group was significantly higher than that of the control group (57.4±42.8 vs. 17.5±12.7, p <.001) (Figure 1A). Patients were classified into 5 subgroups according to their causal drugs, including antibiotics, non-steroidal anti-inflammatory drugs (NSAIDs), histamine-2 receptor blockers (H2- blockers), radiocontrast media (RCM), and others. The others included clopidogrel, proton pump inhibitors, diltiazem, carboplatin, rifampicin, and ethambutol. The most common causal drug was antibiotics (49%), followed by NSAIDs (19%), and H2 blockers (10%). The mean MRGPRX2 levels in patients with DIA caused by antibiotics were higher than in those with other subgroups, but there was no significant difference between the 5 causal drugs (p = 0.964) (Table 1).MRGPRX2 levels of patients with 4–5week time interval were higher (97.4±39.7) than those with 0–1 week (51.9±45.2), 1–2 weeks (54.0±42.1), 2–3 weeks (66.9±39.7), and 3–4 weeks (47.2±27.6) but with no significant difference (p = 0.099) (Table 1). Overall, mean MRGPRX2 levels in each time interval were higher than those in the controls (Figure 1B). There was no significant difference among the frequencies of the causal drugs in each time interval (Table S1).This study demonstrated that serum MRGPRX2 was a potential long-time biomarker for DIA, maintaining high concentration for more than a month from the onset of anaphylaxis. Patients with DIA had higher mean MRGPRX2 levels than controls in all time intervals from the anaphylaxis onset. Thus, MRGPRX2 may be a promising biomarker for supporting a diagnosis of DIA over a longer period than tryptase.This study has limitations in that there was no data of serial changes in MRGPRX2 concentration according to the time interval in the same patient and no direct comparison between MRGPRX2 and tryptase levels. However, this is a pilot study. The performance of MRGPRX2 as a useful biomarker of DIA needs to be further studied using a larger population and in a longer time.

To the Editor,Folliculin, a protein expressed in various types of cells including airway epithelial cells, encoded by the FLCN gene, is associated with the 5′ AMP-activated protein kinase (AMPK) and mammalian target of rapamycin complex 1 (mTORC1) signaling pathways and, it is thought to alter cell-to-cell adhesion and contribute to the pathogenesis of cystic lung disease in Birt-Hogg-Dubé syndrome (1-5). In addition, the geneFLCN regulates the E-cadherin-LKB1-AMPK axis, which controls lung epithelial cell survival and alveoli size (2). In a recent study, serum folliculin levels were found to be higher in patients with asthma than in healthy control groups and high folliculin levels were associated with increased airway hyperresponsiveness in patients with asthma. In vitro data demonstrated the eosinophil-induced release of folliculin from epithelial cells. These clinical and in vitro observations suggest that folliculin may play some role in the interaction between the eosinophils and airway epithelium (6).To investigate the relationship between clinical characteristics and the level of folliculin in asthmatics, the data of a total of 404 patients with asthma and 94 of controls were enrolled and retrospectively reviewed. To correct for the heavily skewed distributions of the serum folliculin levels, values were log-transformed. Study methods, design and definitions used can be found in the online supplement (Study S1).The proportions of males and smokers were significantly higher among the patients with asthma than in the controls, and the mean serum folliculin level in asthmatics was significantly higher than that in controls (4.80 pg/mL versus 4.13 ng/mL; P < 0.001) (Table S1). As the control group was comprised only of males and a significant difference in smoking history was noted, adjusting for sex and smoking history was performed and significantly higher serum folliculin levels were still observed (P < 0.001). We compared the serum folliculin levels between asthmatics and controls subdivided by sex and smoking status. In these subgroups, the serum folliculin levels were still significantly higher in asthmatics than in the control group (Table S2). ROC curve analysis revealed a significant difference in serum folliculin levels between asthmatics and controls (area under the curve = 0.846, confidence interval [CI] 0.80–0.89, P < 0.001); the optimal cut-off value of serum folliculin level that distinguished asthma patients from controls was 4.31 pg/mL after log-transformation, correlating with 83.91% sensitivity and 77.66% specificity (Figure 1). When we perform ROC curve analysis with only the males, the optimal cut-off value of serum folliculin level was 4.33 pg/mL (Figure S1).We compared folliculin levels among the four groups divided by pre- pre-bronchodilator (BD) predicted FEV1 (%) and found a significant difference in serum folliculin level (P < 0.001, Figure S2). Simple and multiple linear regression analysis was performed to determine the correlation between serum folliculin level and lung function in patients with asthma. In simple linear regression analysis, serum folliculin level were significantly correlated with pre-BD FEV1% predicted (β-coefficient = −4.848, P = 0.013), however significance was only marginal after adjusting for age and sex (β-coefficient = −3.199,P = 0.096) in multiple linear regression analysis. This is because, firstly, there was collinearity of folliculin level and age in our data, and secondly, the rate of smokers (85.22%) among males was higher than among females (14.91%), so it seems that lung function in females is higher.Patients with asthma were divided into two groups using the mean value of the logarithmic serum folliculin levels (4.80 pg/mL). Patients in the high-folliculin group were older at the onset of symptoms, heavier smokers and had a significantly lower lung function. The number of acute exacerbations occurring per year was more frequent in the high folliculin group than in the low folliculin group, but no statistical significance was noted (Table 1). When patients with asthma were divided into the upper quartile of folliculin levels and the lower three quartiles combined, those from the high folliculin group in the upper 25 percentile were found to be older and had lower atopy and lung function than the lower folliculin group with the lower 75 percentile combined. (Table S3). Likewise, we also divided the patients into 4 quantile groups according to serum folliculin levels and identified differences in each group in lung function and age (Table S4).A previous in vitro study showed that human airway epithelial cells (HAECs) exposed to leukotriene E4 and peripheral blood eosinophils released folliculin and interleukin (IL)-8, which resulted in the destruction of the integrity of the epithelial cells. The knockdown of folliculin expression resulted in a decrease in IL-8 release and suppression of epithelial cell activation, which restored the epithelial integrity in HAECs. In their study, folliculin was suggested to be associated with a higher serum transforming growth factor-β1 level, which was associated with worsening of airway inflammation and remodeling (6,7). Consistent with theses result, a higher serum folliculin level in patients with asthma than in healthy controls was also observed in our study, in addition, we showed that an increase in serum folliculin level was associated with a decrease in basal lung function. As folliculin is released from bronchial epithelial cells in response to compressive stress that mimics a bronchospasm (8, 9), we postulate that chronic airway inflammation produces mechanical stress on the airway epithelium, thereby inducing oxidative damage and release of folliculin with changes in the epithelial cell structure. Therefore, we assume that folliculin is associated with the airway inflammation and remodeling pathway in patients with asthma. In our study, serum folliculin level showed no association with serum laboratory variables, suggesting that the increase in folliculin level following mechanical stress is independent of other serum inflammatory markers.In conclusion, our study demonstrates for the first time that serum folliculin concentration is higher in patients with asthma, and it is associated with worse lung function independent to other serum inflammatory markers. Thus, folliculin may represent a novel biomarker related to lower pulmonary function in patients with asthma and further studies are warranted to evaluate the mechanism and test our hypothesis.Keywords: asthma; folliculin; biomarker

Background Although genome-wide association studies (GWAS) represent the most powerful approach for identifying genes that influence asthma, no studies have established the genetic susceptibility to asthma in the Korean population. To identify genetic variants associated with adult Korean asthmatics and compare them with the significant single nucleotide polymorphisms (SNPs) of UK asthmatics from UK Biobank. Methods Asthmatic patients were defined as having asthma if they were diagnosed by a doctor or taking medications for asthma. Controls were defined as having no asthma and chronic obstructive pulmonary disease. We performed the quality controls, genotype imputation, GWAS, and PrediXcan analysis. In GWAS, P value < 5×10-8 is considered significant. We compared significant SNPs between Korean asthmatics and UK asthmatics. Results A total of 1,386 asthmatic patients and 5,205 controls were analyzed. The SNP rs1770, located near human leukocyte antigen (HLA)-DQB1, was the most significant SNP (P=4.5×10-10). In comparison with 24 SNPs in GWAS of UK asthmatics, 6 SNPs were significant with the same odds ratio direction, including signals related to type 2 inflammation (e.g., IL1RL1, TSLP, and GATA3) and mucus plugging (e.g., MUC5AC). HLA-DQA1 showed an opposite odds ratio direction. HLA-DQB1 gene demonstrated significant imputed mRNA expression levels for lung tissue and whole blood. Conclusions The SNP rs1770 of HLA-DQB1 was the most significant SNP in Korean asthmatics. There were similarities and discrepancies in genetic variants between Korean and UK asthmatics. The GWAS of Korean asthmatics should be replicated and compared with those of GWAS of other ethnicities.