Zein, Joe

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

Introduction Despite the identification of multiple susceptibility loci by genome-wide association studies (GWAS), considerable chronic obstructive pulmonary disease (COPD) heritability remains unexplained. Aim To identify interaction networks of airway epithelial cell DNA methylation in COPD and further explore potential correlations with airway bacterial composition, as potentially collective regulators of biological pathways influencing COPD severity. Methods Using DNA isolated from bronchial airway brushings of 67 ever-smokers (>20 pack-years) from the SubPopulations and InteRmediate Outcomes Measures in COPD Study (SPIROMICS), we assessed proportion of DNA methylation (β) by epigenome-wide association study (EWAS) and examined associations of differentially methylated CpG probes (DMPs) with risk for moderate-to-severe COPD (N=34) versus absent or mild COPD (N=33). We tested co-methylation modules generated by Weighted Correlation Network Analyses (WGCNA) for associations with moderate-to-severe COPD and with bacterial genus-level relative abundances (16S rRNA sequencing). Results EWAS-identified nominally significant DMPs enriched for lung function GWAS loci. Eigengenes in six WGCNA modules were associated with moderate-to-severe COPD (false discovery rate <0.05). Four of those modules were enriched for forced expiratory volume in 1 s/forced vital capacity GWAS loci, and five overlapped with DMPs from EWAS. Overlapping CpG loci in three COPD-associated modules were adjacent to mucin genes; one had 10 genes highly ranked by connectivity with MUC5B, including important pathway genes: B3GNT6 , DGKI and ITGA8 . CpGs in an independent COPD-associated module showed the most correlations with Rothia , with directionality suggestive of negative associations with moderate-severe COPD. Conclusions Bronchial epithelial DNA methylation modules enriched for lung function GWAS loci associate with COPD severity in SPIROMICS. Potential module relationships to bronchial bacterial composition require further validation.

Abstract RATIONALE. Significant sex-differences exist in asthma across the lifespan. Asthma prevalence and severity are greater in pre-pubescent boys compared to girls but greater in women after puberty. In women, asthma may improve after menopause when sex hormones wane. To address potential mechanisms underlying these changes, we compared the airway transcriptomics signatures of post-menopausal vs. premenopausal women. METHODS. We used bulk RNA seq data from bronchial epithelial cells from three different cohorts (SARP III, GEO-GSE85567, GEO-GSE201955) to perform a comprehensive analysis of differential gene expression and regulation. Data were combined into a meta-analysis, to compare pre-menopausal (n=114 cases with asthma, 48 controls) to post-menopausal women (n=52 cases with asthma, 13 controls). Differential gene expression between pre- and postmenopausal women was analyzed separately for asthma (Figure 1A) and control groups (not shown). Significant differentially expressed genes (DEGs) were identified by contrasting effect sizes (log fold-changes) between asthma and control groups using a Z-score interaction test. Accordingly, a scatter plot (Figure 1B) was created to represent whether differential gene expression between pre- and post-menopausal conditions is modified by disease status (asthma vs. control). The scatter plot also highlights that these findings are related to asthma per. se. and not to normal aging and menopausal transition. Pathway enrichment analysis was conducted using the differential expression data, followed by differential regulation analysis of the identified pathways. Statistical significance was assessed using a false discovery rate (FDR) threshold of 5%. RESULTS. Out of 15,468 genes that passed quality control, 19 genes were differentially expressed in bronchial epithelial cells from pre- and post-menopausal women with asthma (as compared to controls) (Figure 1A). Of these, 5 genes were down-regulated (ITLN1, POSTN, FCRL3, CCL22, PCSK6), and 14 genes were up-regulated (SPP1, S100A9, A2ML1, TNFRSF10C, CSF3R, OLFM1, C1QL2, NLRP3, HAL, PI3, RNASE1, TMEM178B, TNNI3, RNF175) when comparing pre- and post-menopausal women with asthma (Figure 1A). Of these 19 DEGs, 14 were differentially expressed in pre- as compared to post-menopausal women with asthma, but not in controls (Figure 1B). Comparing pre- to post-menopausal women with asthma, biologic pathways related to the innate and adaptive immune system activation and mitochondrial function (Electron transport Chain oxidative phosphorylation and ATP production) were both differentially regulated and expressed. CONCLUSIONS. This analysis highlights several DEGs and pathways that characterize asthma pathogenesis when comparing pre- and post-menopausal women, and emphasize the role of female sex hormones on the immune system response and mitochondrial function in asthma pathogenesis.

Abstract Rationale: MUC5B and MUC5AC are neighboring genes on chromosome 11p15 that encode the two predominant mucins secreted in the respiratory airways. Increased airway mucin concentrations and an imbalance between these two mucins drive COPD symptoms and the development of airflow obstruction or COPD, but the epigenomic regulation of this loci has not been characterized in the airways. Methods: We isolated DNA from bronchial airway brushings of 72 participants with &gt;20 pack-years smoking history from the Subpopulations and Intermediate Outcomes Measures in COPD Study (SPIROMICS). 36 participants had GOLD-based moderate-to-severe COPD (FEV1/FVC&lt;0.70, FEV1&lt;80%) and 36 were without or with mild COPD. We used the Illumina Infinium Methylation EPIC BeadChip Array to assess proportion of DNA methylation (β) spanning 689,937 CpG sites. We evaluated proportional DNA methylation (β) at 96 CpG loci within a 103kB region using matrixQTL for cis-expression quantitative trait methylation (eQTM: 10kB flanks relative to MUC5AC/MUC5B) integrating bulk RNA-seq data and mQTL models integrating gene sequencing data to identify DNA variation associated with proportion eQTM methylation. Models included age, sex, BMI, clinical site, current smoking vs past smoking, total pack-years, three ancestral components of ancestry, chipset, and position. Results: We identified a top eQTM, cg00524486, marginally associated with MUC5B expression (coefficient estimate=8.75, punadjusted=0.0014, pFDR=0.065, Figure 1B). This eQTM was associated with CT scan parametric response mapping-based functional small airways disease (PRM-fSAD, beta coefficient[β]=3.64, p=0.011). We tested for DNA variation associated with methylation of this top eQTM which identified two methylation quantitative trait loci (mQTL: rs885454 [β=0.044, minor allele frequency[MAF]=0.09, pFDR=0.01], rs868902 [β=0.020, MAF=0.43, pFDR=0.08, Figure 1A) associated with cg00524486 methylation, of which the more frequent mQTL, rs868902, was marginally associated with PRM-fSAD (p=0.07) in the larger SPIROMICS cohort with MUC5B DNA sequencing data. Conclusions: This preliminary multi-omic study of the lower airway epithelium demonstrates that the expression of MUC5B is epigenetically regulated by neighboring CpG probe methylation (eQTM) represented by DNA variation (mQTL). These studies require further confirmation in a larger dataset of airway samples and support a role for epigenetic variation as a biologic contributor and biomarker of early COPD pathogenesis.

Abstract Background. Sarcopenia (i.e. skeletal muscle loss) and physical frailty are significant contributors to adverse clinical outcomes in lung cancer patients. Nutritional intake and physical activity influence skeletal muscle mass and strength. We determined the impact of lung cancer on clinical outcomes in hospitalized patients with sarcopenia, frailty, or sarcopenia+frailty. Design. The Nationwide Inpatient Sample (NIS) (years 2018-2020) from the United States was analyzed to study adult hospitalized patients with a diagnosis of lung cancer. Sarcopenia and frailty were defined by ICD10 codes that have been previously validated. Multivariate regression models were generated to determine the clinical impact of sarcopenia, frailty, or sarcopenia+frailty in patients with a diagnosis of lung cancer as compared to a general medical population (GMP) without lung cancer. Age was restricted to &gt;40 years old for both cohorts because lung cancer is rare in those less than 40 years old. Models were adjusted for age, race, sex, and comorbidities (cardiovascular disease, anemia, coagulopathy, HIV, depression, diabetes mellitus, and COPD). Results. Compared to the GMP (n=126,099), patients with lung cancer (n=241,913) were older (mean+SD age: 69.6+10.0 vs. 67.6+13.3 years, p&lt;0.001), and had a higher prevalence of sarcopenia (22.5% vs 9.0%, p&lt;0.001), frailty (53.4% versus 49.8%, p&lt;0.001), and sarcopenia+frailty (15.3% versus 6.8%, p&lt;0.001). COPD was common and found in 52.3% of lung cancer patients and 20.2% of the GMP. In lung cancer patients, the most common medical reasons for admission were sepsis, pneumonia, and COPD exacerbation. In GMP patients, the most common medical reasons for admission were sepsis, congestive heart failure, and acute kidney injury. In lung cancer patients, frailty had the greatest association with mortality (unadj OR 3.12, 95% CI: 3.02-3.23; adj OR 3.04, 95% CI: 2.91-3.17). Sarcopenia+frailty was most associated with greater healthcare utilization including greater healthcare cost (unadj OR 1.31, 95% CI: 1.29-1.32; adj OR 1.28, 95% CI: 1.26-1.30) and length of stay (unadj OR 1.57, 95% CI: 1.55-1.58; adj OR 1.50, 95% CI: 1.48-1.52). Sarcopenia+frailty were more likely to require additional healthcare facilities upon discharge and were the least likely to be discharged to home (unadj OR 0.32, 95% CI: 0.32-0.33; adj OR 0.35, 95% CI: 0.33-0.36). (see Table 1). Conclusion. While frailty was most associated with in-hospital mortality in lung cancer patients, sarcopenia+frailty was most associated with greater healthcare utilization. Our findings highlight the need to develop targeted interventions to improve clinical outcomes in lung cancer patients with sarcopenia and/or frailty.

Abstract RATIONALE: Significant sex differences exist in the prevalence and severity of asthma across the lifespan. Asthma is more prevalent and severe in pubescent boys compared to girls but worse in girls and women after puberty. The transition to menopause brings significant reductions in estrogen and progesterone levels. In this study, we test the hypothesis that the effect of menopause on asthma prevalence and outcomes is modified by the use of female sex hormones. METHODS: We used data from nine two-year cycles (1999-2000 to 2015-2016) from NHANES to determine the association between menopause and asthma outcomes. We defined severe asthma based on GINA treatment classes 4&5. We used analytic sample weights to compute national estimates according to NHANES guidelines. Adult (age &gt;18 years) women were considered to be menopaused if (1) they reported to be menopaused during the NHANES survey, (2) they had bilateral oophorectomy, or (3) they were ≥52 years. Weighted logistic regression analyses were used to adjust for age, race, body mass index (BMI), smoking, blood eosinophil count, female hormone therapy, and oral contraceptives. Analyses were performed using the SURVEY package in R. RESULTS: The cohorts’ clinical characteristics are listed in table 1. Compared to pre-menopause (weighted n=60,394,897), menopause (weighted n=52,364,594) was associated with higher rates of active asthma (9.2 vs. 8.9%, p=0.004) and severe asthma (5% vs. 1.8%, p&lt;0.001), but not lifetime asthma (15.1 vs. 16.3%, p=0.063). Notably, postmenopausal women had higher BMIs (weighted median [IQR]: 28.1 [24.2-33.1] vs. 26.1 [22.6-32.1], p&lt;0.001), longer asthma durations (weighted median [IQR]: 20.0 [7.0-41.0] vs. 15.0 [8.0-24.0], p&lt;0.001), and lower lung functions (p&lt;0.001) (table 1). Adjusted analysis did not show significant associations between menopause and lifetime asthma (adjusted OR, 1.074; 95% CI, 0.901-1.280), active asthma (adjusted OR, 1.072; 95% CI, 0.865-1.328), or severe asthma (adjusted OR, 1.369; 95% CI, 0.958-1.954). Interestingly, a history of female hormone therapy and oral contraceptives use was associated with a higher risk of lifetime asthma (adjusted OR, 1.191; 95% CI, 1.031-1.375 and adjusted OR, 1.287; 95% CI, 1.169-1.417 respectively). Among women with active asthma, menopause was not associated with non-severe exacerbations (adjusted OR, 1.335; 95% CI, 0.883-2.021) or with severe exacerbations requiring ER visits (adjusted OR, 1.416; 95% CI, 0.818-2.451). Conclusion: The increased prevalence of active asthma and severe asthma among menopausal women can largely be attributed to age-related changes, comorbidities, and hormone therapy use rather than menopause alone.

Abstract RATIONALE: Women are more likely to develop asthma after puberty, and their symptoms can fluctuate during the menstrual cycle, pregnancy, and menopause. Menopausal women with asthma face unique challenges, as hormonal fluctuations during this stage can impact respiratory health. About one-third of women undergo hysterectomy before age 60, with most procedures occurring before natural menopause. While half of these surgeries preserve the ovaries, the other half include oophorectomy, creating distinct hormonal environments. Despite the traditional view of the uterus as hormonally inactive, there remains a critical gap in our understanding of the potential impact of hysterectomy on respiratory health.METHODS: We used data from nine two-year cycles (1999-2000 to 2015-2016) from NHANES to determine the association between hysterectomy and asthma phenotypes. Severe asthma was defined based on GINA treatment classes 4&5. Analytic sample weights were used according to published NHANES guidelines to create national estimates. Asthma status (lifetime, active, severe) was obtained for women without (weighted n=90,348,593) or with a history of hysterectomy (weighted n=22,310,898). Weighted logistic regression analyses were used to determine the impact of hysterectomy on asthma prevalence, severity, exacerbations, and asthma-related emergency room (ER) visits adjusting for age, race, body mass index (BMI), smoking history, blood eosinophil count, a history of unilateral or bilateral oophorectomy, female hormone therapy, and oral contraceptive use. Analyses were performed using the SURVEY package in R.RESULTS: Clinical characteristics of women with and without a history of hysterectomy are listed in Table 1. Overall, hysterectomy was associated with higher asthma prevalence (18.1 vs. 15.1%) and severity (5.1 vs. 2.9%). For all women, hysterectomy was associated with a higher risk for lifetime asthma (adjusted OR, 1.262; 95% CI, 1.1079-1.477) but not for active asthma (adjusted OR, 1.146; 95% CI, 0.93-1.412) or severe asthma (adjusted OR, 0.996; 95% CI, 0.748-1.325). Interestingly, oophorectomy was associated with a higher risk for lifetime asthma (adjusted OR, 1.228; 95% CI, 1.102-1.368 for unilateral; adjusted OR, 1.232; 95% CI, 1.232-1.513 for bilateral) and active asthma (adjusted OR, 1.417; 95% CI, 1.232-1.629 for unilateral; adjusted OR, 1.507; 95% CI, 1.143-1.986 for bilateral). Among women with active asthma, hysterectomy was associated with severe exacerbations requiring ER visits (adjusted OR, 1.845; 95% CI, 1.157-2.941) but not with non-severe exacerbations (adjusted OR, 1.176; 95% CI, 0.811-1.705).CONCLUSIONS: This study highlights the influence of hysterectomy on asthma risk and severity and emphasizes the role of female hormones in asthma pathogenesis.