Ortega, V E

Abstract Rationale Sleep is an important dimension of COPD symptomology. Poor self-reported sleep quality has been independently associated with increased exacerbation risk and worse quality of life. There is limited evidence around the longitudinal sleep quality of patients with smoking-related lung disease. Methods Using the SPIROMICS longitudinal cohort of heavy smokers, we used group-based trajectory modeling to understand sleep quality over time as measured by serial retests of the Pittsburgh Sleep Quality Index (PSQI). We considered all participants of Black or Non-Hispanic White race who completed the PSQI in Visits 1-4, a span of 3 years. We considered race, gender, and social environmental exposures as time-stable risk factors for trajectory group membership. We adjusted for age, lung function, mood symptoms and self-reported COPD symptom severity were considered as time-varying factors. We selected the number of trajectory groups by minimizing Bayesian Information Criteria and polynomial order by maximizing the significance of exponential terms. For quality assurance we calculated average posterior probability of correct group assignment. Calculations were performed by complete case analysis and multiple imputation with chained equations. Results We analyzed 2,838 participants yielding 76,153 usable observations. Overall, 39.2% of participants had complete observations for all variables of interest at every measured visit, and 60.8% had at least one relevant observation missing. The largest single source of missingness in our dataset was the comorbidity count, for which 44% of participants had a missing observation. We identified two major trajectory groups. In one group, the PSQI was persistently high with little variation over time, containing 77.7% of the overall study population. The second group had low PSQI at baseline but experienced wide fluctuation over time. Risk factors for persistently poor sleep included female gender, Black race, and more adverse social conditions at either the neighborhood or individual level (Table). The average posterior probability of correct group assignment was 94.6% among the Low Variable group and 89.5% among the Persistent High group. Results were similar by complete case and imputed analysis, save that BMI did not independently predict group membership in the latter. Conclusion Historically underserved groups are at long term risk for poor sleep outcomes in smoking-related lung disease, even among those who are highly motivated and participate in clinical research. More work is needed to ensure equitable access to sleep medicine in order to improve the quality of care in COPD. This abstract is funded by: Nina Ireland Program for Lung Health

Abstract Background Regulator of G protein signaling (RGS)2 terminates bronchoconstrictive Gaq signaling, and reductions in RGS2 expression associate with a hypercontractility phenotype. RGS2 promoter variants rs2746071 and rs2746072 are hypofunctional in gene reporter assays, and individuals with severe asthma homozygous for these two RGS2 variants experience higher asthma exacerbation rates. Whether these RGS2 promoter variants directly cause a hypercontractile human airway smooth muscle (HASM) phenotype is unknown. Methods We used the hTERT immortalized HASM cell line, derived from a human non-asthmatic donor. RGS2expression was reduced with siRNA (150nM anti-RGS2 vs. control siRNA) or through CRISPR transformation (Synthego©) to express the RGS2 promoter variants of interest, confirmed by Sanger sequencing. Gene and protein expression were quantified by qPCR and Western blotting. Intracellular Ca ++ flux and oscillation was measured with Fluo-8 (Abcam) with excitation/emission at 490/525 nm. HASM contractility to histamine treatment was measured using Traction Force Microscopy implemented in a 96-well plate format. Results Relative to cell lines with reference genotypes, HASM CRISPR transformed with the two RGS2 promoter variants exhibited a 41.8% (95% CI 28.6%--62.5%; p < 0.0001) reduction in RGS2 relative expression by qPCR, and a 35% (95% CI 8.6%—85.7%; p = 0.024) reduction in protein expression by Western blot. Methacholine-triggered calcium flux was nearly 50% greater for the cell lines with the RGS2 homozygous variants vs. those with reference genotypes (p = 0.043) as was the contractile response to histamine. HASM with reference genotypes exposed to anti-RGS2 siRNA showed the same phenotype, consistent with a causal relationship between the variants and expression (decreased) and receptor function (enhanced). Similarly, HASM CRISPR-transformed with the two RGS2 promoter variants exhibited an up to 3-fold increase in contraction magnitude (represented by strain energy, reported in pJ) in response to histamine >1uM (p < 0.05) relative to cell lines with reference genotypes. Conclusion Two promoter single nucleotide polymorphisms substitutions are sufficient to reduce RGS2 expression and induce a hypercontractile HASM phenotype. This abstract is funded by: AAAAI Bridge Award, NHLBI R21

Abstract Rationale We hypothesize that corticosteroid resistance causes uncontrolled inflammation and severe asthma. The genetic and molecular mechanisms underlying steroid responsiveness in asthma remain poorly understood (PMID28493293). This study aims to characterize transcriptomic profiles distinguishing steroid responders from steroid resistant asthma. Methods Longitudinal bulk RNA-seq of SARP3 sputum samples was analyzed before and after an “induced phenotype” with 40 mg triamcinolone acetonide (TCA) injection (PMID 27967215). Participants were classified as responders or non-responders based on whether their FEV₁ change after TCA exceeded the median. Paired differential gene expression analyses were performed separately for each group using limma and voom, with participant as a factor and adjustment for sputum cell count differentials. Z-scores were calculated to compare gene expression changes after TCA injection between responders and non-responders, and pathway enrichment was assessed using the fgsea package. Results Baseline demographics and clinical characteristics of 696 SARP3 asthma participants receiving TCA are shown in Table 1. The median [IQR] FEV1 change post-TCA was 1.81% [-2.16, 7.17]. Responders had higher baseline FEV1%, FEV1/FVC ratio, blood eosinophil counts, and sputum eosinophil percentages, but lower serum CRP levels. Steroid responsiveness was associated with asthma severity (Table 1). Among 154 participants with paired sputum RNA-seq data, 70 (45%) exhibited >1.81% improvement in FEV₁ and were classified as responders. Following TCA treatment, non-responders showed no significant changes in gene expression. In responders, 18 genes were upregulated and 203 downregulated. Downregulated genes were associated with Th2 immunity (IL13, IL1RL1, CCL17, SIGLEC8), IgE signaling (FCER1A, FCER2), epithelial barrier function and mucus production (MUC2, MUC7, ALOX15, TFF3), tissue remodeling and fibrosis (MMP10, TIMP3), ciliary function (DNAH5, CFAP43, FOXJ1), and airway hyperreactivity (HRH4, RAMP1). Key genes notably downregulated in responders compared to non-responders included IL13, CCL5, PTGDR2, FCER2, and CLEC10A. Pathway analysis revealed enhanced IL-10-mediated anti-inflammatory signaling, suppression of pro-inflammatory pathways (NF-κB, IL-1, Th1/Th2, asthma, arachidonic acid metabolism, chemokine, NOTCH4), and regulation of mitochondrial oxidative phosphorylation. Conclusions Steroid resistance impairs glucocorticoid-mediated suppression of pro-inflammatory pathways, including NF-κB, IL-1, and Th1/Th2 signaling. This persistent airway inflammation limits improvements in lung function and contributes to worse clinical outcomes, including increased airflow obstruction, and symptom burden. Glucocorticoid resistance in asthma may be driven by receptor dysfunction, airway inflammation, epigenetic changes, and environmental exposures resulting in persistent airway inflammation and remodeling. Elucidating the molecular mechanisms underlying steroid resistance is critical for developing personalized targeted therapies in asthma. This abstract is funded by: NIH-NHLBI - R01 HL161674 (PI: Joe Zein)

Abstract Rationale Primary ciliary dyskinesia (PCD) pathogenic gene variants disrupt motile cilia, leading to both PCD and asthma through epithelial dysfunction. Asthma shows sex differences, and progesterone regulates ciliary beat frequency, indicating that genetic defects in ciliary proteins may exert sex-dependent effects. Therefore, we hypothesize that rare, pathogenic variants in PCD genes contribute to asthma risk in a sex-specific manner. Methods We performed a rare-variant aggregation analysis of the DNAH11 gene using UK Biobank OQFE exome dosages, with sex-stratified analyses. Within each sex, genotype dosages were coded as individual-level minor allele counts (0-2) and filtered to include variants with a minor allele frequency (MAF) ≤1% and a minor allele count (MAC) ≥5 across the cohort. Asthma phenotypes were defined from a curated dataset (N = 452,927), excluding COPD and other lung diseases. For each sex, logistic null models adjusted for age were fitted, and gene-level associations for binary outcomes were tested using SKAT, Burden, and SKAT-O. To assess variant-specific influences, we performed leave-one-out (LOO) analyses, sequentially omitting each variant and recalculating p-values. The effect size across the cohort was calculated by fitting a Firth logistic regression comparing carriers to non-carriers, adjusted for age by sex. Finally, sex modification was tested using a Firth model including carrier-by-sex interaction term. Results After filtering, DNAH11 included two rare variants; 7:21564185-G>T and 7:21591370-G>A in each sex (MAF≈1-2x10-5). Among females (N = 245,718; 28,649 with asthma), 16 carriers were observed (MAC≈16). Gene-based tests showed a significant association with asthma (Burden_p=0.004; SKAT-O_p=0.005; SKAT_p=0.013). Firth carrier model OR was 4.58 (95% CI 1.62-11.98, p = 0.0055) (Figure 1A). LOO analyses (Figure 1B) showed both variants contributed to the signal; removing 7:21591370-G>A increased SKAT-O p to 0.075 (Δ-log10p≈+1.15), while removing 7:21564185-G>T increased p to 0.014 (Δ-log10p≈+0.41, with the association remaining significant after removing the latter. Among males (N = 207,209; 20,777 with asthma), 10 carriers were observed (MAC≈10) with no significant association (Burden_p=0.926; SKAT-O_p=0.797; SKAT_p=0.668; Firth_p=0.781). The carrier-by-sex interaction term in the combined Firth model was not significant (p = 0.190), suggesting the female-specific signal could not be detected in the males due to limited power. Conclusions This analysis provides primary evidence that these rare, pathogenic variants in the DNAH11 PCD gene may contribute to asthma susceptibility in a sex-specific manner, with a significant association observed only in females. The signal was predominantly driven by 7:21591370-G>A. This suggests that genetic disruption of ciliary function may interact with female-specific hormonal factors (i.e., progesterone) to influence asthma risk. This abstract is funded by: NIH

Abstract Rationale Asthma and obesity are common comorbidities, both driven by inflammation and oxidative stress. Oxylipins are derivatives of polyunsaturated fatty acids that regulate the onset and resolution of inflammation. Oxylipin profiles are altered in obesity and have been shown to differentiate asthma from non-asthma; however, the association between oxylipins and asthma outcomes and the impact of obesity is not known. This study aims to identify differences in serum oxylipins associated with asthma morbidity and evaluate the effect modification by BMI. Methods We analyzed 150 participants in the Best African American Response to Asthma Drugs (BARD) clinical trial. Ninety oxylipins were quantified using liquid chromatography tandem mass spectroscopy from serum obtained at the time of randomization. Oxylipins found to be above the limit of detection in > 80% of samples were log-transformed and included in the analysis. Asthma severity measures included past 12-month systemic corticosteroid use for asthma exacerbation, spirometry, asthma control and asthma quality of life questionnaires (AQLQ). The associations between oxylipins and asthma outcomes were tested using multivariable regression models adjusted for sex, age, BMI, absolute eosinophil count, percent African ancestry, and child vs adult/adolescent cohort. A false discovery rate (FDR) of < 0.1 is used to determine statistical significance. Results Of the 90 oxylipins measured, 42 (46.7%) were detected in > 80% of all samples and included in the analysis. In multivariable models, the pro-inflammatory arachidonic acid (AA) derivative 5,6-DiHETrE was associated with decreased odds of past 12-month systemic corticosteroid use (FDR-adjusted p-value 0.0628). AA derivatives TXB2, PGF2a, and 6-α-prostaglandin were associated with increased FEV1/FVC (FDR-adjusted p-values=0.0925). Thirteen oxylipins were associated with decreased asthma-related quality of life in multivariable models, including AA metabolites 5S-HETE, 5-KETE, 14,15-EET, LTB4, and Lipoxin A4; dihomo-gamma-linoleic acid metabolites 5-HETrE and 15-HETrE; linoleic acid metabolites 3-OxoODE and 9-OxoODE; and eicosapentaenoic acid metabolites 15-HEPE and 17,18-EpETE, all with FDR-adjusted p-values<0.05. Oxylipin-BMI interaction was significant only in the AQLQ models. In all but LTE4, increased concentrations of oxylipins were associated with a decreased quality of life in individuals with higher BMI (Figure). Six of these oxylipins are products of lipoxygenase activity. Conclusions In this study, we find decreased quality of life with increasing concentrations of pro-inflammatory and resolving oxylipins among those with obesity. These oxylipins are primarily produced via lipoxygenases and may represent an important inflammatory pathway in obesity-associated asthma. Quantification of sputum oxylipins and correlation with serum levels will be important in understanding potential mechanisms and is forthcoming. This abstract is funded by: K12TR004412, R01HL123385, U10HL098075, Parker B. Francis Fellowship, 2L30HL159725

Abstract Rationale Asthma, a chronic inflammatory airway disease, can be influenced by hormonal fluctuations that affect immune function and airway responsiveness. During in-vitro fertilization (IVF), women undergo sequential hormonal treatments—including gonadotropin administration to stimulate follicle development and induce ovulation, and progesterone and estrogen to support implantation and early pregnancy. These precisely timed hormonal changes may affect asthma symptoms and increase the risk of exacerbations. This study evaluates the hypothesis that undergoing IVF increases the risk of asthma exacerbations prior to, but not during or after pregnancy. Methods Data were extracted from the Mayo Clinic electronic health records. Women were included if their pregnancy was recorded in the Mayo Clinic obstetric delivery dataset. Asthma was defined as the presence of an ICD code J45.xx in combination with a prescription for any asthma medication. For women with multiple pregnancies, only the first pregnancy was analyzed. An asthma exacerbation was defined as a course of oral corticosteroids lasting ≤28 days or a corticosteroid injection. Multivariable logistic regression was used to assess exacerbation risk, adjusting for age, body mass index (BMI), and prior history of diabetes mellitus. Results Among 3,623 pregnant women, 112 underwent IVF treatment. Compared with non-IVF patients, women undergoing IVF were older (mean ± SD: 34.16 ± 5.24 vs. 29.11 ± 5.55, p < 0.001), had lower parity (1.44 ± 0.79 vs. 1.83 ± 1.20, p = 0.001), and fewer full-term deliveries (1.31 ± 0.79 vs. 1.68 ± 1.11, p = 0.001) (Table 1). There were no differences in pre-pregnancy BMI, racial distribution, ethnicity, or prior history of diabetes mellitus. Adjusted analysis indicated that IVF was associated with an increased risk of asthma exacerbation in the year before pregnancy (adjusted OR [95% CI]: 1.097 [1.025-1.173]), but not during (adjusted OR [95% CI]: 1.044 [0.973-1.121]) or after pregnancy (adjusted OR [95% CI]: 1.026 [0.963-1.094]). Conclusion These findings suggest that the preconception hormonal changes associated with IVF may temporarily worsen asthma control. Further studies are warranted to replicate these results, clarify the underlying mechanisms, and guide asthma management in women undergoing fertility treatment. This abstract is funded by: NIH

Abstract Rationale Asthma is a chronic inflammatory airway disorder influenced by pulmonary and systemic factors. Epidemiologic and experimental data suggest associations between asthma, anemia, and reduced iron stores. Sex may modify this relationship: females, with lower baseline iron reserves, menstrual blood loss, and hormone-mediated immune effects, may exhibit greater susceptibility, whereas males, with higher iron stores and androgen-related anti-inflammatory influences, appear less affected. This study tested the hypothesis that diminished iron stores, rather than anemia itself, are associated with asthma, and that this relationship differs by sex. Methods Data were analyzed from nine consecutive NHANES 2-year cycles (1999-2018), a nationally representative U.S. dataset. Lifetime asthma was defined as a prior physician diagnosis and active asthma as “Do you still have asthma?”. Anemia was defined by WHO criteria (hemoglobin <12 g/dL in females and <13 g/dL in males), and iron deficiency as ferritin <30 ng/mL. Accordingly, iron status was categorized as normal, iron deficiency anemia (IDA), iron deficiency without anemia (IDWA), and anemia without iron deficiency (AWID). Analyses followed NHANES guidelines and were conducted with the SURVEY package in R. Results Female participants were older and had lower eosinophil counts, ferritin, and hemoglobin levels, with a higher prevalence of anemia, lifetime asthma, and current asthma. Similar trends were observed among participants with available ferritin data (Table 1). In unadjusted analyses, ferritin levels were inversely associated with lifetime (β = -0.19; p <0.001) and active asthma (β = -0.35; p <0.001), but these associations were not significant after adjustment for sex, age, BMI, race, and log10-transformed eosinophil count (lifetime: β = 0.03; p = 0.61; active: β = -0.08; p = 0.40). In contrast, anemia was associated with increased odds of lifetime (adjusted OR [95% CI], 1.52 [1.21-1.92]) and active (1.50 [1.13-1.99]) asthma in males, but not in females (0.98 [0.86-1.12] and 0.98 [0.83-1.15], respectively). Among males, AWID was associated with active asthma (2.07 [1.08-3.96]), whereas IDA and IDWA were not. No associations were observed among females. Conclusion The relationship between anemia and asthma may be primarily driven by inflammation related to asthma rather than true iron deficiency. After adjustment, ferritin was not independently associated with asthma, whereas anemia—particularly AWID—remained associated among males. These findings suggest that asthma-associated systemic inflammation contributes to anemia of chronic disease, underscoring the need to distinguish inflammatory anemia from nutritional iron deficiency in research and clinical management. This abstract is funded by: NIH

Abstract Rationale Asthma contributes substantially to healthcare burden and morbidity through frequent exacerbations. Emerging evidence suggests that genetic factors play a crucial role in modulating susceptibility to asthma exacerbations. To better understand the genetic architecture of exacerbations, we conducted a genome-wide association study (GWAS) in the Tapestry cohort. Methods The Tapestry Study, a large-scale, decentralized and clinical grade genomic sequencing study, conducted Exome+® sequencing at Helix, Inc. on 98,075 Mayo Clinic participants. After excluding patients with ≥10 pack-years of smoking and/or emphysema, we performed a GWAS of asthma exacerbations in 3,960 adults with asthma based on ICD codes. Cases (n = 1,059) were defined as individuals who experienced at least two exacerbations between 2020 and 2025; the remaining 2,901 individuals served as controls. Asthma exacerbations were defined by oral or systemic corticosteroid prescriptions with durations ranging from 3 days up to 28 days. Logistic regression models adjusted for age, sex, BMI, and the first six principal components of ancestry. Replication was performed in 1,273 asthma patients (439 African Americans and 834 non-Hispanic Whites) from the NHLBI-sponsored Severe Asthma Research Program (SARP 1-3), with adjustments for age, sex, BMI, and the first 10 principal components of ancestry. Results Based on a minor allele frequency (MAF) threshold of > 0.05, we analyzed 5,256,135 genome-wide variants. Genome-wide significant associations for asthma exacerbations (p < 5.0 × 10⁻8) included loci at a pseudogene in SMPD4P1 (rs680548, MAF = 0.39, OR = 1.37, p = 9.10 × 10⁻9). Additional variants showing suggestive associations included loci within ENDOD1 (rs638776, OR = 0.69, p = 1.20 × 10⁻6), PCSK2 (rs2206454, OR = 1.32, p = 3.53 × 10⁻6), RBFOX1 (rs17667084, OR = 0.60, p = 5.11 × 10⁻6), and CDC42EP3 (rs721660, OR = 1.29, p = 5.68 × 10⁻6). Subthreshold associations in Tapestry including loci at CDC42EP3 (rs4670213, OR = 1.27, p = 1.41x10-5, MAF = 0.36), and SDR39U1 (rs12587663,OR = 0.83, p = 5.86x10-4, MAF = 0.46) were replicated in the SARP longitudinal visit 5 with p-values of 8.523x10-3 (rs4670213, OR = 1.78), and 2.88x10-4 (rs12587663,OR=0.43), respectively. Conclusion Our GWAS for asthma exacerbations in the Tapestry cohort identified variant loci relevant to mitophagy mechanisms (CDC42EP3, PMID: 40783597), immune signaling and RNA splicing regulation (RBFOX1, PMID: 31301374), and neuromodulatory pathways (PCSK2, PMID: 32794589), all previously implicated in asthma pathophysiology. Collectively, these findings highlight potential novel genetic contributors to asthma exacerbations and provide a foundation for improving risk stratification and therapeutic targeting in asthma management. This abstract is funded by: This abstract is funded by: Mayo Clinic Funding: X01 from the NHLBI for TOPMed whole-genome sequencing and NIH grants R01 HL142992, HL69116, HL69167, HL69170, HL69174, U10 HL109164, U10 HL109257, U10 HL109146, U10 HL109172, U10 HL109250, U10 HL109250, U10 HL109250, U10 HL109168, U10 HL109152, U10 HL109086, U10 HL109046.

Abstract Rationale Higher African genetic ancestry has been associated with lower lung function, among individuals from specific racial-ethnic groups. The influence and interactions of genetic variation and ancestry in lung function is poorly understood. This study investigates ancestry-associated loci influencing lung function via admixture mapping and fine-scale allelic association. Methods 512 African American patients with asthma (≥12 years) from the Severe Asthma Research Program and Collaborative Study on the Genetics of Asthma (SARP/CSGA) cohorts were genotyped on the OmniExpress and HumanHap1M arrays or had whole-genome sequencing (WGS). For admixture mapping, global ancestry was inferred using ADMIXTURE and local ancestry estimated using RFMix v1.5.4 with CEU (N = 99) and YRI (N = 108) references across 15,095 local ancestry segments. Peak regions and contiguous segments (p < 10-3) were fine mapped in SARP using local ancestry estimated from WGS with MAF ≥0.03. The Local Ancestry Adjusted Allelic Association (LAAA) model was used to fine map local genetic effect, local ancestry effect, and the interaction between the two, adjusted for age, sex, height, BMI, cohort, and percentage global African ancestry. SNPs with local genetic or interaction p < 10⁻³ were tested for replication in 282 adolescent/adult participants from the Best African Response to Asthma Drugs (BARD) cohort genotyped with the MEGA array imputed to TOPMed (MAF ≥0.03). Results Mean African genetic ancestry was 81.3% in SARP and 80.1% in BARD. In SARP, African genetic ancestry was inversely associated with lung function (FEV₁:β = −7.9, p = 0.005; FVC:β = −9.0, p = 0.004), with stronger effects in females (FEV₁: β = −9.3, p = 0.003; FVC: β = −11.4, p = 0.001) than males (FEV₁:β = −6.4, p = 0.237; FVC:β = −5.7, p = 0.340). Similar total and sex-specific associations were identified in BARD. Admixture mapping in SARP/CSGA identified significant peaks for maximum FVC (p < 1.87 × 10⁻4) on chromosomes 8 and 17 near RP1 and RBFOX3, and near-significant peaks on chromosome 5 near IQGAP2, SNRPCP2, SV2C and for the maximum FEV₁/FVC ratio on chromosome 10 near PCDH15. Fine mapping identified the top SNP on chromosome 8 (rs73587686; p-allele-dose=9.0x10-5; RGS20; q11.23), with 62 additional SNPs overlapping RGS20. SNP rs7825816 was also identified in SARP (β = 2.35, p-allele-dose =1.03 × 10⁻4) and replicated in BARD (β = 1.15, p-allele-dose =0.03). In BARD, LAAA also identified eight additional SNPs (p ≤ 0.05) in the same LD block (chr8:53914976-53918297) overlapping with RGS20, ATP6V1H, and TCEA1. Conclusions We identified multiple chromosomal regions associated with lung function in asthma patients of predominantly African genetic ancestry. Lung function associations were strongest at genetic variation near a key that was replicated in an independent asthma cohort. This abstract is funded by: R01 K08 HL118128, R01 HL142992, R01 NR013700, R01 HL69167, R01 HL161674, HL69116, HL69167, HL69170, HL69174, U10 HL109164, U10 HL109257, U10 HL109146, U10 HL109172, U10 HL109250, U10 HL109250, U10 HL109250, U10 HL109168, U10 HL109152, U10 HL109086-04, U10 HL098115, HL098102, HL098096, HL098075, HL098090, HL098177, HL098098, HL098107, HL098112, HL098103, and K12 HL89992