Introduction: Pulmonary hypertension (PH) frequently occurs in individuals with chronic obstructive
pulmonary disease (COPD), impacting their prognosis. Understanding the genetic background of COPD
PH could enhance disease classification and treatment decisions. We aimed to evaluate known pathogenic
variants of genes associated with pulmonary arterial hypertension (PAH) in a large cohort of smokers.
Methods: We analyzed whole genome sequencing and Phase 1 clinical data from the Genetic
Epidemiology of COPD (COPDGene) cohort, consisting of non-Hispanic white and African-American
smokers with and without COPD, as part of the Trans-Omics in Precision Medicine (TOPMed) program.
We used WGSA v0.95, integrating outputs from ANNOVAR, VEP, SnpEff, and ClinVar, to annotate 12
genes with definitive evidence (BMPR2, ACVRL1, ATP13A3, CAV1, EIF2AK4, ENG, GDF2, KCNK3, KDR,
SMAD9, SOX17, TBX4), 3 genes with moderate evidence (ABCC8, GGCX, TET2), 6 genes with limited
evidence (AQP1, BMP10, FBLN2, KLF2, KLK1, PDGFD), and 5 genes with disputed evidence (BMPR1A,
BMPR1B, NOTCH3, SMAD1, SMAD4) for association with PAH according to ClinGen. We assessed the
prevalence of different classes of pathogenic variants and the clinical and chest CT imaging
characteristics of variant carriers. Results: We analyzed 10,550 whole genomes and 133,060 intronic and
exonic variants in previously identified PAH genes. Only 2,256 variants (1.7%) were reported in ClinVar,
with 1,501 classified as benign/likely benign, 724 as variants of uncertain significance (VUS) or with
conflicting interpretations, and 31 as pathogenic/likely pathogenic. We identified pathogenic/likely
pathogenic heterozygous variants in 6 genes with definitive evidence (14 variants in 19 subjects),
including 5 pathogenic variants in EIF2AK4 carried by 6 subjects. Carriers of pathogenic variants,
including those in genes with moderate or disputed evidence (36 subjects, representing 0.3% of the
COPDGene cohort), showed no differences in demographic factors, lung function, or pulmonary vascular
imaging phenotypes compared to carriers of VUS (5,279 subjects) or benign variants (4,845 subjects) in
both univariable or models adjusted for age, sex, race, and genetic ancestry. Conclusion: Clinically
annotated pathogenic variants in known PAH genes were identified in the COPDGene cohort. Cross
sectional analysis revealed no substantial clinical differences between carriers and non-carriers of these
variants. Future research will incorporate non-ClinVar variants and leverage multi-omics and longitudinal
data for a more comprehensive understanding of these genetic influences

Introduction:
Mucus plugs in smokers with and without COPD are associated with worse lung function and may represent an important treatable trait in airways disease. While genetic risk factors have been identified for symptomatic syndromes associated with mucus plugging (e.g. bronchiectasis), genetic risk factors for CT-identified mucus plugs, which are often asymptomatic, have not been identified.

Methods:
We performed a whole genome sequencing association study of mucus plugs in 3,433 non-Hispanic white (NHW) and 1,021 African American (AA) individuals in the Genetic Epidemiology of COPD (COPDGene) using data generated from the Trans-Omics in Precision Medicine (TOPMed) study. We calculated lung mucus plugging scores using chest CT scans. We analyzed a dichotomized overall score after adjusting for age, gender, smoking pack-years, current smoking status, sequencing center, and genetic ancestry. We also performed a secondary analysis using the value in each lobe. We investigated genome-wide single variants, loss-of-function rare variants within genes and candidate associations in genes with reported associations for related phenotypes (cough, phlegm, bronchiectasis). We considered a P-value of 5x10-8 for genome-wide significance. We also used SomaScan 1.3k data in a subset of 518 individuals to investigate the association between the trait and proteins.

Results:
We found no significant associations in the single variant, rare variant, and protein analyses using the binary score for whole mucus plugging. Our top findings in single variant associations were near FAM80B and SLC2A13 (P = < 2 x10-7). In rare variant analysis, the top association for high-confidence loss-of-function rare variants was in HAL (P = 3.84x10-6), while our top protein association was CXCL13 (P = 2.29x10-4). Notably, we did not identify any associations with previously described associations with cough, phlegm, or in other candidate genes associated with bronchiectasis. In a secondary analysis of lobar mucus plugging binary scores, we identified 4 genome-wide significant regions; our top finding was near ZNF407 (8.02x10-9) with other less significant associations near ZNF816, CCSER1, and L3HYPDH.

Conclusions: In a whole genome sequencing and proteomic study of mucus plugging, we did not identify genome- or proteome-wide significant associations; nor did we find associations in a set of candidate genes and variants. In a secondary analysis, we did identify associations with lobe-specific values, though their clinical significance remains unclear. Future work will include analysis of quantitative phenotypes and in additional samples.

Abstract:
To investigate the impact of time varying nature of DNA methylation (DNAm) on the progression of chronic obstructive pulmonary disease
(COPD), we conducted a longitudinal epigenome-wide association study (EWAS). The EWAS findings were integrated with proteomic data to reveal
the interplay between methylation and proteomics in COPD. Methods: Infinium MethylationEPIC BeadChip data was obtained from peripheral
blood leukocyte DNA from 4,493 current and former smokers from the Genetic Epidemiology of COPD (COPDGene) study consisting of two time
points of DNAm and FEV1 data (baseline and 5-year follow-up visits) of non-Hispanic white and African American subjects with a history of
smoking, a median age of 62.2 years at enrollment and 51 percent male subjects. SomaScan blood proteomic data was available from the 5-year
follow-up visit. Linear mixed-e"ects regression (LMER) was used to find the associations between longitudinal DNAm and longitudinal forced
expiratory volume in 1 second (FEV1); adjusted for sex, age, race, cell counts, pack-years of smoking, current smoking status, height and time
since baseline visit. Causal mediation analysis to identify proteins potentially mediating the relationship between DNAm and FEV1 was conducted
using a nonparametric bootstrap procedure from the R package Mediation. Functional overlap analysis on the genome-wide significant CpGs was

performed using the eFORGE tool. Results: From the longitudinal FEV1 and longitudinal DNAm data, LMER identified 66 CpGs having genome-
wide significant associations with FEV1 (p ≤ 5 x 10-8). Intraclass correlation (ICC) of the 66 CpGs calculated using DNAm M-values from both

time points revealed that the majority (40) have ICC between 0.5 and 0.8 and 22 CpGs have ICC greater than 0.8, suggesting that FEV1-
associated CpGs are stable over 5 years. Chromosome 17 demonstrated three significant associations over a 440 base pair region annotated to
the SOCS3 gene (cg13343932 with p = 2.12 x 10-18, cg11047325 with p = 8.77 x 10-16 and cg18181703 with p = 1.07 x 10-13). An X
chromosome CpG near the zinc-finger repressor, BCOR, was also associated with longitudinal FEV1 (cg18422972 with p = 2.14 x10-8).
Functional Overlap analysis of the 66 CpGs revealed significant enrichment in histone methylation (H3K4me1 and H3K4me3) in blood and lung
tissue. Mediation analysis identified ITPRIPL1, NOTCH1 and IGFBP5 as three candidate proteins partially mediating the signal between DNAm and
FEV1. Conclusions: EWAS of longitudinal DNAm data revealed that the CpG sites that are stable over time are associated with longitudinal lung
function (FEV1) and are linked with epigenetic and inflammatory signals.