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Abstract RATIONALE: Chronic Obstructive Pulmonary Disease (COPD) is a leading cause of mortality worldwide. We previously published the polygenic transcriptome risk score (PTRS) which uses the cumulative effect of predicted gene expression to construct genetic predictors for complex diseases, and we demonstrated the value of PTRS for COPD built on Genotype-Tissue Expression (GTEx) Lung tissue with significantly improved cross-ancestry portability. However, the postmortem collection and lack of lung disease status on GTEx samples likely affect results. We hypothesized that the performance of PTRS will be improved by using disease-relevant expression quantitative trait loci (eQTLs) to construct the score. Here, we aimed to improve performance of PTRS by leveraging disease-relevant eQTLs. METHODS: We constructed two transcriptome prediction models, Elastic Net (EN) and Prediction Using Models Informed by Chromatin conformation and Epigenomics (PUMICE), using TOPMed lung RNA-seq data from the Lung Tissue Research Consortium (LTRC), which included COPD patients undergoing lung surgery. We compared their performance with two published models built on RNA-seq from GTEx Lung tissue, GTEx-EN and GTEx-PUMICE. We first integrated each of four transcriptome models with multi-ancestry GWAS of FEV1/FVC ratio (Shrine et al. 2023) to generate transcriptome-wide association study (TWAS) results, which produced trait-associated genes. Pathway analysis was then conducted on significant TWAS genes. Finally, the PTRS was computed on TWAS genes that were included in the top 20 significantly enriched pathways. We tested performance of PTRS in race-stratified analysis for moderate-to-severe and severe COPD in COPDGene. RESULTS: Our models produced more significant TWAS genes (FDR<0.05) and had more overlaps with genes identified for FEV1/FVC ratio from Shrine et al. 2023 compared to two GTEx models (Fig. A). Additionally, our models had significantly higher AUC for predicting both moderate-to-severe and severe COPD in race-stratified analysis. The mean AUC from LTRC-EN for predicting severe COPD was 0.57 and 0.55 respectively for EUR and AFA, which was significantly higher than mean AUC of 0.54 and 0.51 from GTEx-EN (Delong p-value=8.15x10-10 for EUR and 1.42x10-3 for AFA) (Fig. B). Similarly, the PTRS derived from our models produced higher Odds Ratios (OR) per standard deviation. The mean OR from LTRC-PUMICE for moderate-to-severe COPD was 1.25 and 1.18 respectively for EUR and AFA, while it was 1.17 and 1.12 from GTEx-PUMICE (Fig. C). CONCLUSIONS: Our study demonstrates the value of leveraging disease-relevant RNA-seq to construct transcriptome prediction models and their improvement on identifying lung function genes and predicting COPD across ancestries.

Abstract Rationale: The club cell secretory protein (CC16), encoded by the SCGB1A1 gene, has an anti-inflammatory role in airways diseases, including asthma (Li, AJRCCM 2023; Voraphani, AJRCCM 2023). We hypothesize that additional pathway genes regulated or co-expressed with CC16 have cumulative effects on asthma severity outcomes by modulating airway inflammation and maintaining lung homeostasis. Methods: As published previously, four genes were downregulated in CC16 knockout experiments and correlated with CC16 levels: BPIFA1, SFTPD, LTF, and LYZ (Iannuzo, Front. Immunol 2023). We developed a four-gene biomarker score in 94 patients with asthma from the NHLBI-sponsored Severe Asthma Research Program (SARP3) who underwent research bronchoscopy for lower airway epithelial brushings, RNA isolation, and bulk RNA sequencing. We log-transformed the raw values of each transcript; obtained standardized residuals from regression models that included sex, age, and batch effects; and summed the standardized residuals of all four proteins to estimate a standardized sum score (Zhai, JACI Allergy 2024). Logistic regression models tested the four-gene score, three-gene score without LTF, and individual transcripts for associations with exacerbations during 12-month follow-up and zero-inflated negative binomial regression for number of exacerbations. Results: 30.85% percent of 94 patients experienced at least one exacerbation at the end of follow-up. The CC16-associated four-gene biomarker score showed protective effects on asthma exacerbation risk with a 54% percent reduction in exacerbation risk for every one-fold higher score (OR=0.46, 95% CI: 0.27-0.78, p =0.0040, Figure 1). Transcript expression of BPIFA1 (OR=0.87, 95% CI:0.66-1.16, p=0.337), SFTPD (OR=0.33, 95% CI:0.04-2.52, p=0.283), LYZ (OR=0.62, 95%CI:0.28-1.39, p=0.248), and LTF (OR=0.36, 95% CI:0.18-0.74, p=0.0053) all indicated protective directionality with respect to exacerbation risk. However, only LTF demonstrated a statistically significant association. When we excluded LTF, the remaining three-gene score showed a trend toward association with risk (OR=0.64, 95% CI:0.40-1.03, p=0.068) and was significantly associated with number of exacerbations (RR=0.49, 95% CI:0.26-0.95, p=0.034). Compared to LTF alone (OR=0.48, PPV=0.50, NPV=0.71, Sensitivity=0.14, AUC=0.65), the combined four-gene score (OR=0.46, PPV=0.60, NPV= 0.73, Sensitivity=0.21, AUC=0.68) showed improved predictive performance. Conclusions: We identified a CC16-associated transcriptomic predictive score for asthma exacerbations, with LTF showing the strongest effect. Although the other three genes showed individually weak and non-significant associations with exacerbations, the three gene score was significant for number of exacerbations.We demonstrate the potential of this transcriptomic score as a predictive biomarker for asthma severity-related outcomes. Further research investigating these genes and their interaction with other CC16-associated immune pathways could provide insight into therapeutic targets.

Abstract Rationale Bronchial hyperreactivity (BHR) is a hallmark of asthma, frequently presenting as exercise-induced dyspnea in children and adolescents. Identifying BHR is essential for accurate diagnosis and management of exercise symptoms. The methacholine challenge test (MCT) is the gold standard for diagnosing BHR due to its high sensitivity in detecting airway hyperresponsiveness. However, the MCT requires specialized equipment and stringent protocols, which can limit its availability and use in some clinical settings. An alternative approach, the bronchodilator response test (BDR), is more accessible and assesses the reversibility of airway obstruction following bronchodilator administration. BDR testing is a routine component of asthma diagnostics for evaluating airflow limitation, making it a potentially useful tool for BHR assessment. However, the ability of BDR testing to reliably detect BHR in exertional dyspnea is still uncertain. Prior research has shown that BDR testing, while specific, has low sensitivity for BHR when compared to the MCT, raising questions about its standalone utility in clinical practice. The objective of this study was to evaluate the diagnostic performance of the BDR in detecting bronchial hyperreactivity in exertional dyspnea, using the MCT as the reference standard. By analyzing sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), we aimed to assess whether BDR testing could serve as a reliable alternative to MCT for diagnosing BHR. Methods We conducted a retrospective study of pediatric patients who underwent cardiopulmonary exercise testing, BDR, and MCT as part of their assessment for exertional dyspnea from July 2020 to February 2023. Patients up to 18 years of age were included. A positive BDR was defined by a 10% or greater improvement in FEV1% or FVC%, while a positive MCT was defined by a 20% reduction in FEV1%. Data are presented in a 2x2 table, with the MCT as the reference standard. Results and Conclusion In a cohort of 98 patients, 20 underwent both bronchodilator and methacholine testing. The BDR test, using MCT as the reference, showed a sensitivity of 16.7%, specificity of 87.5%, PPV of 66.7%, and NPV of 41.2%. These results suggest that, due to its low sensitivity, BDR testing alone is insufficient for BHR detection when clinical suspicion of asthma is high. The MCT remains the preferred diagnostic method, though using both tests may provide a more comprehensive evaluation of airway reactivity in children presenting with exercise symptoms.

Abstract Rationale Exertional dyspnea in children presents a complex diagnostic challenge, often requiring multiple evaluations to uncover underlying etiologies. Exercise testing is the standard for diagnosing exercise-induced bronchoconstriction (EIB) in children but requires significant resources. Fractional exhaled nitric oxide (FeNO), a marker of airway inflammation, has been proposed as an easier alternative to predict the need for exercise testing. The aim of this study is to evaluate the diagnostic utility of fractional exhaled nitric oxide (FeNO) as a non-invasive marker of bronchial hyperreactivity in pediatric patients with exertional dyspnea, by assessing its sensitivity, specificity, and predictive values in comparison to cardiopulmonary exercise testing (CPET), the gold standard for diagnosing exercise-induced bronchoconstriction (EIB). This study seeks to determine whether FeNO can reliably guide the need for CPET, potentially streamlining the diagnostic process in children with suspected EIB, while also identifying any limitations of FeNO as a standalone diagnostic tool. Methods In this retrospective study, we reviewed pediatric patients up to 18 years of age who presented with unexplained dyspnea and underwent both CPET and FeNO testing from July 2020 to February 2023. A post-exercise decline in FEV1% of ≥10% during CPET defined a positive result for EIB. For FeNO, a threshold of ≥25 parts per billion (ppb) was considered indicative of bronchial hyperreactivity. Diagnostic agreement between FeNO and CPET was analyzed using a 2x2 table, with CPET as the reference standard. Results Among 98 patients who completed CPET, the cohort had a mean age of 14 years (SD ± 2.42), mean BMI of 22.2 kg/m² (SD ± 5.25), and 71% were female. Of these, 40 patients also underwent FeNO testing. Compared to CPET, FeNO exhibited a sensitivity of 100%, a specificity of 2.6%, a positive predictive value of 5.1%, and a negative predictive value of 100% for identifying bronchial hyperreactivity in this setting. Conclusion FeNO testing shows high sensitivity but limited specificity for detecting bronchial hyperreactivity, effectively ruling out but not confirming it independently of CPET. These findings reinforce the essential role of CPET in diagnosing EIB and suggest that FeNO may serve best as an adjunctive tool, rather than a primary diagnostic measure, in evaluating children with exertional dyspnea. A limitation of this study is the reliance on a single FeNO threshold, which may not account for individual variability in bronchial inflammation responses.

Abstract Rationale Exertional dyspnea in children often necessitates a thorough evaluation to determine potential causes, with bronchial hyperresponsiveness (BHR) and exercise-induced bronchoconstriction (EIB) being among the most common. Cardiopulmonary exercise testing (CPET) is widely recognized as the gold standard for diagnosing EIB; however, it requires significant patient cooperation and specialized resources. The methacholine challenge test (MCT), a bronchial provocation test, is commonly employed to evaluate baseline airway hyperreactivity. Nonetheless, its reliability in predicting EIB in pediatric patients experiencing exertional dyspnea has not been well established. This study aims to evaluate the diagnostic accuracy of MCT compared to CPET in detecting BHR among pediatric patients with unexplained dyspnea. This assessment will help determine the potential of MCT as a complementary diagnostic tool for EIB. Methods We conducted a retrospective cohort study involving pediatric patients (up to 18 years of age) who underwent both CPET and MCT as part of their clinical evaluation for exertional dyspnea from July 2020 to February 2023. A reduction of ≥20% in FEV1% was considered a positive result for the methacholine challenge. In the CPET, a decrease of ≥10% in FEV1% post-exercise was indicative of EIB. The results from both tests were compiled in a 2x2 table, using MCT as the reference standard. Results Out of the 98 patients who underwent CPET, 53 also completed a methacholine challenge test. The mean age of participants was 14 years (SD ± 2.42), with a mean BMI of 22.2 kg/m² (SD ± 5.25), and 71% of the patients were female. Using CPET as the reference standard, the MCT demonstrated a sensitivity of 66.7%, specificity of 58%, positive predictive value of 8.7%, and a negative predictive value of 96.7% for identifying BHR in patients with exertional dyspnea. Conclusions MCT exhibited moderate sensitivity and specificity, and a high negative predictive value, indicating its effectiveness in ruling out BHR in children with exertional dyspnea. However, it is less reliable as a standalone diagnostic tool for EIB, and CPET remains essential for an accurate diagnosis of EIB in these patients.

Abstract Primary ciliary dyskinesia (PCD) results from mutations in over 50 genes that affect motile cilia (PMID: 29905515). PCD incidence is at least 1:7600 (PMID: 35051411). Asthma may be a PCD comorbidity (PMID: 36431268); and antigen stasis on the airway surface in PCD may predispose to asthma (PMID: 3831866). Methods. We analyzed the association between PCD and asthma in children ≤ 18 years old in two large electronic health record (EHR)-based databases: the Indiana Network for Patient Care Research (INPCR) cohort of 20 million EHRs and, in a validation cohort, TriNetX, with 112 million EHRs. We needed to address two issues. First, there is no ICD 10 code for PCD. Secondly, clinicians may confuse asthma and PCD in assessment. Therefore, we analyzed two diagnoses that, together, are unlikely to occur together in the absence of PCD: bronchiectasis (ICD10:J47) and situs inversus totalis (Q89.3) together (B-SIT). We asked whether subjects with B-SIT also had an asthma diagnosis ( ICD10 J45). We matched cases to controls, 1:3 in INPCR; and 1:1 in TriNetX, controlling for age, sex and race. We used conditional logistic regression adjusted for the matching strategy. For INPCR, we performed a conditional exact test: numbers were too small for adjusted models. This study was approved by the Indiana University and Case Western Reserve University IRBs. Results. In the initial INPCR study, 100% of children with B-SIT had an asthma diagnosis (p < 0.001). In the TriNetX validation cohort (Matched 1:1, n = 115 each), 84 children with B-SIT had asthma and 31 did not; in the controls, 12 had asthma and 103 did not (p = 2.2 x 10-16). The adjusted OR for B-SIT patients having asthma was 22.26 (95%CI: 10.80 – 45.91; p <0.001). Discussion. In two large EHR data sets, we found that most children with likely PCD (B-SIT) had a diagnosis of asthma. Local antigen stasis in PCD could contribute to the asthma phenotype in PCD, or perhaps even to asthma among patients heterozygous for PCD genes (PMID: 38457400). Additional studies are needed to understand the immunology, and this study is that it will need to be validated prospectively using airway reactivity testing after drug with-hold. In summary, children with PCD may need evaluation for airway reactivity. Further, providers should be alert to whether there are cardinal features of PCD among any of the children cared for with a diagnosis of asthma.

Abstract Introduction Unilateral pulmonary vein atresia (UPVA) is a rare congenital anomaly involving absent or obstructed pulmonary veins (PV), often leading to chronic pulmonary venous stasis, recurrent infections, and hemoptysis. Although typically diagnosed in infancy, UPVA can present in adulthood. This report presents a 63-year-old male referred as a case of chronic thromboembolic pulmonary disease (CTEPD) who was diagnosed with UPVA. We describe the diagnostic complexities and potential explanation for loss of pulmonary artery (PA) perfusion in such cases. Case Presentation A 63-year-old man was referred to rule out CTEPD. His past medical history included coronary artery disease and sleep apnea. He presented with chronic cough, repeated hemoptysis, recurrent pneumonias requiring prolonged antibiotics use, and progressive exertional dyspnea. Physical examination was unremarkable. Pulmonary function tests showed moderate restriction and moderately-reduced diffusion capacity. A ventilation-perfusion (V/Q) scan revealed complete absence of perfusion in the left lung. [figure1] High-resolution CT revealed a straight left atrial border sign with absent left superior and inferior PVs, left pulmonary arterial tree hypoplasia, hypoplastic left lung with fibrotic changes due to chronic venous-lymphatic engorgement and pulmonary hypoperfusion. CT also showed compensatory right lung hyperinflation and bronchial artery collaterals. Echocardiography indicated an atrial septal defect (ASD) with no pulmonary vascular disease. Based on CT angiography, UPVA was considered the leading diagnosis. Discussion UPVA is a congenital disease rarely presenting in adulthood with recurrent infections, hemoptysis, and fibrosis. It often accompanies congenital cardiac conditions, like ASD in our case. Pulmonary angiography may reveal ipsilateral PA hypoplasia and atrophied peripheral vessels. The absence of perfusion in the V/Q scan could be explained by reversed blood flow in the PA from congestion caused by the atretic PVs and ipsilateral backflow from the bronchial arteries collaterals. Differential diagnosis like CTEPD was excluded due to atypical complete loss of perfusion of the left lung, and lack of the other radiological stigmata. Swyer-James-MacLeod syndrome was excluded due to a mismatched perfusion defect. Proximal Interruption of the PA was also excluded due to normally developed proximal left and right PAs. Imaging is crucial in showing absent PVs and hypoplastic ipsilateral lung. Conservative management for UPVA is recommended. Given recurrent infections and hemoptysis, pneumonectomy can be considered. Conclusion This case illustrates the atypical presentation of UPVA as a CTEPD mimicker, presenting with absent pulmonary perfusion unilaterally, which can be explained by reversed pulmonary flow secondary to venous engorgement and backflow from bronchial artery collaterals. Figure1:

Abstract RATIONALE: Endothelial-to-mesenchymal transition (EndMT) is a cellular process in which endothelial cells (ECs) transition into mesenchymal cells, a mechanism recently linked to pulmonary vascular remodeling in pulmonary hypertension (PH). However, the regulatory mechanisms driving EndMT in PH remain largely undefined. MicroRNA-153 (miR-153), a small non-coding RNA, regulates gene expression by targeting the 3'-untranslated region (3'-UTR) of mRNA, affecting mRNA translation and stability. This study investigates miR-153's role in modulating EndMT in PH. METHODS: Human lung vascular endothelial cells (LVECs) were isolated from healthy individuals and patients with idiopathic pulmonary arterial hypertension (IPAH). In vitro, normal LVECs were exposed to hypoxia (3% O₂) for 72 hours or treated with TGF-β1 (10 ng/mL) for 7 days to induce EndMT. Levels of miR-153 and EndMT markers were quantified by qPCR, Western blotting, or immunostaining. Proliferation and apoptosis of ECs were assessed using EdU and TUNEL assays, respectively. LVECs were transfected with miR-153 inhibitor or mimic, then exposed to normoxic or hypoxic conditions, respectively for 72 hours. In vivo, mice were subjected to either hypoxia (10% O₂ for 6 weeks, Hx-PH) or Sugen5416/hypoxia (20 mg/kg i.p. plus 10% O₂ for 6 weeks, SuHx-PH) to induce PH. The PH phenotype in mice was evaluated by hemodynamic measurements and histological analysis of H&E-stained lung tissues. RESULTS:In silico analysis identified miR-153 as a direct regulator of human SNAI1 and SNAI2 genes via binding to their 3'-UTR. Downregulation of miR-153 in IPAH LVECs was correlated with elevated SNAI1/2 expression, increased EndMT, and higher proliferation compared to normal LVECs. Hypoxia or TGF-β1 treatment reduced miR-153 expression, increased EndMT transcription factors (SNAI1/2), mesenchymal markers (SM-22, vimentin), and reduced EC markers (CD31, VE-cadherin). In normoxic LVECs, miR-153 inhibition significantly upregulated SNAI1/2, increased EndMT and EC proliferation, and reduced apoptosis. Conversely, miR-153 mimic abolished hypoxia- and TGF-β1-induced EndMT. Overexpression of miR-153 attenuated hypoxia-induced EC survival. In PH mouse models, reduced miR-153 levels were associated with activated SNAI1/2, EndMT, and pulmonary vascular remodeling. CONCLUSIONS: Downregulation of miR-153 drives phenotypic transition of LVECs to proliferative mesenchymal cells via SNAI-mediated EndMT, contributing to pulmonary vascular remodeling and PH. Therefore, miR-153 could be a potential therapeutic target for PH treatment.

Abstract Rationale Patients with pulmonary hypertension (PH) often require significant assistance from caregivers. Caregivers frequently help patients with numerous PH-related tasks, such as medication administration and insurance coordination, in addition to emotional and other forms of support. However, little is known about optimal strategies for engaging and empowering caregivers in PH. We sought to improve understanding of PH patients’ and caregivers’ perspectives on the role of caregiving to identify ways to better support them. Methods We performed a cross-sectional survey of PH patients and/or their adult caregivers participating in the Pulmonary Hypertension Association conference in August 2024. Questions focused on roles and perspectives towards caregiving. Study participation was voluntary without monetary or other compensation. Results Sixteen patients and 18 caregivers completed the survey, with 9 patient and caregiver pairs. Patient characteristics and responses are detailed in Table 1. The majority of patients (87.5%) reported that caregivers play a “very important” role in their lives. (Table 1) Most surveyed caregivers were patients’ significant others (10/18, 55.5%) or parents (7/18, 38.9%), with average age 59 years, and were employed full-(10/18, 55.5%) or part-time (3/18, 16.7%). Regarding their role(s), all caregivers (18/18) reported providing emotional support, 7/18 (38.9%) physical support (e.g. mobility, hygiene assistance), 7/18 (38.9%) help with medications, and 13/18 (72.2%) help with transportation to appointments. 4/18 (22.2%) caregivers reported they spend more than 20 hours/week on caregiving-related tasks while most reported they spend 5-20 hours/week. Caregivers detailed challenges regarding emotional support (e.g. “Spouse seems to be very short with me most of the time. I think she hates this disease and takes it out on others”), among others. However, they also reported caregiving to be rewarding (e.g. “Seeing my husband improve, and getting to be on his side to advocate for him and other patients.”) Caregivers had many suggestions for what they would like to see in a program focused on supporting them, including “Online education such as resources and experts such as new treatments, insurance information…How to advocate better. Tips for self care” and “Connection to therapist who understand chronic diseases and the challenges of long term caregivers.” Conclusions Caregivers play an integral and multifaceted role in the lives of patients with PH. Caregivers would appreciate additional education, resources and support.

Abstract Rationale: Since the initial descriptions of pulmonary arterial hypertension (PAH), there has been a significant increase in PAH diagnoses among the geriatric population. Studies suggest that older PAH patients exhibit unique hemodynamic profiles and treatment responses. We hypothesized that patients ≥80 years old would have a unique clinical profile and worse outcomes. Methods: The Pulmonary Hypertension Association Registry (PHAR) is a prospective cohort of PAH and chronic thromboembolic pulmonary hypertension (CTEPH) patients new to care at a Pulmonary Hypertension Care Center in the US. Individuals <18 years of age and those with CTEPH were excluded. Clinical data, quality of life measures, and functional capacity were recorded at time of enrollment and at follow up visits. Octogenarians were defined as those ≥80 years old and non-octogenarians as <80 years old. Associations between age subgroups with the change in six-minute walk distance (6MWD), and worsened WHO functional class (WHO FC) at the next visit, and time to all-cause mortality were assessed using linear, binomial, and Cox regression, respectively. Results: The study sample included 87 PAH octogenarian patients and 2,583 non-octogenarian patients at enrollment. The Table shows that octogenarian PAH patients were of similar sex and race distribution as others, but had lower body mass index. Octogenarians were more likely to have idiopathic or CTD-related PAH, and differed from younger patients in terms of several sociodemographic variables. PAH treatment, WHO FC, quality of life, and hemodynamic also differed between the groups. Octogenarians had a lower change in 6MWD (-26m, 95%CI -52 – -2, p = 0.066) and worse WHO FC (2.0, 95%CI 1.1 – 3.8, p = 0.038) over time compared to other adults after adjustment for sex, BMI, PAH etiology, annual income, marital status, right atrial pressure, mean pulmonary artery pressure, and PAH treatment. Octogenarians had an increased risk of death compared to younger adults (hazard ratio 1.7, 95%CI 1.1 – 2.6, p = 0.022) after adjustment for covariates. Conclusions: While the eldest PAH patients may have some comparable features to younger patients, they are different in a variety of socioeconomic variables, disease type, hemodynamics, and number of PAH therapies. The eldest PAH patients demonstrate worse exercise and functional capacity with time and a 70% increased risk of death compared to younger patients. Further studies are needed to better characterize PAH in the elderly, as this may lead to more effective treatments and enhanced quality of life.