Blood DNA methylation patterns are highly predictive of prenatal exposure to smoking, and differential methylation has been associated with maternal alcohol use. We extended this to determine whether DNA methylation patterns in cord blood are associated with prenatal exposure to opioids, cannabis, and polysubstance use. We also evaluated whether DNA methylation patterns have predictive utility. We examined 932 mother-child pairs in the Boston Birth Cohort between 1998 and 2020 with cord blood DNA methylation data. For each substance self-reported within 72 hr after birth, we performed an adjusted linear regression analysis at 865,859 CpG sites to identify related methylation differences. We generated polyepigenetic scores using summary statistics for each exposure and assessed predictive ability using cross-validation and receiver operating characteristic curves. Specificity of methylation associations was evaluated by assessing overlap across exposure summary statistics and using logistic regression for methylation scores, adjusted for concurrent use. We identified methylation changes at 72, 21, and 1 CpG suggestively associated with prenatal exposure to opioids, cannabis, and polysubstance use, respectively (p < 1e-6), in cord blood, reported for the first time for these exposures. We identified two loci associated at epigenome-wide significance with opioids and one with polysubstance use (p < 1e-8). Methylation scores were highly predictive and exposure-specific, with area under the curve accuracy of 91% for opioids, 90% for cannabis, and 93%-98% depending on polysubstance number. These CpGs provide biologic insights for reducing the impact of substance exposure, and these findings may serve as a biomarker of prenatal substance exposure for future studies and potential clinical utility.

Van der Woude syndrome (VWS) is an autosomal dominant disorder characterized by lower lip pits and orofacial clefts (OFCs). With a prevalence of ∼1 in 35,000 live births, it is the most common form of syndromic clefting. Most VWS is attributed to variants in IRF6 (∼70%) or GRHL3 (∼5%), leaving up to 25% of individuals without a molecular diagnosis. Both IRF6 and GRHL3 function in a transcriptional regulatory network (TRN) governing differentiation of periderm, a single epithelial cell layer preventing pathological adhesions during palatogenesis. Periderm disruption can elicit a spectrum of phenotypes, including lip pits and OFCs, pterygia, and severe or fatal congenital anomalies. Understanding these mechanisms is vital in improving health outcomes for individuals with peridermopathies. We hypothesized genes encoding members of the periderm TRN, including kinases such as atypical protein kinase C (aPKC) acting upstream of IRF6, could harbor variants resulting in VWS. Consistent with this hypothesis, we identified 7 de novo variants (DNs) and 11 rare variants in PRKCI in 18 individuals with clinical features of syndromic OFCs and peridermopathies. Among the identified DNs, c.1148A>G (p.Asn383Ser) was found in five unrelated individuals, indicating a hotspot mutation. We functionally tested 12 proband-specific alleles in a zebrafish model. Three alleles, c.389G>A (p.Arg130His), c.1148A>G (p.Asn383Ser), and c.1155A>C (p.Leu385Phe), were confirmed loss-of-function variants. We also show that phosphomimetic Irf6 can rescue the effects of aPKC inhibition, supporting placement of PRKCI within this TRN. In summary, we identified PRKCI variants as causative for VWS and syndromic OFC with other features of peridermopathies.

Background/Objectives: Maternal exposures are known to influence the risk of isolated cleft lip with or without cleft palate (CL/P)—a common and highly heritable birth defect with a multifactorial etiology. Methods: To identify new risk loci, we conducted a genome-wide gene–environment interaction (GEI) analysis of CL/P with maternal smoking and vitamin use in Filipinos (Ncases = 540, Ncontrols = 260). Since GEI analyses are typically low in power and the results can be difficult to interpret, we applied multiple testing frameworks to evaluate potential GEI effects: a one degree-of-freedom (1df) GxE test, the 3df joint test, and the two-step EDGE approach. Results: While no genome-wide significant interactions were detected, we identified 11 suggestive GEIs with smoking and 24 with vitamin use. Several implicated loci contain biologically plausible genes. Notable interactions with smoking include loci near FEZF1, TWIST2, and NET1. While FEZF1 is involved in early neuronal development, TWIST2 and NET1 regulate epithelial–mesenchymal transition, which is required for proper lip and palate fusion. Interactions with vitamins encompass CECR2—a chromatin remodeling protein required for neural tube closure—and FURIN, a critical protease during early embryogenesis that activates various growth factors and extracellular matrix proteins. The activity of both proteins is influenced by folic acid. Conclusions: Our findings highlight the critical role of maternal exposures in identifying genes associated with structural birth defects such as CL/P and provide new paths to explore for CL/P genetics.

BACKGROUND:Orofacial clefts (OFCs) are the most common craniofacial birth defects, affecting 1 in 700 births, and have a strong genetic basis with a high recurrence risk within families. AIMS:While many of the previous studies have associated common, noncoding genetic loci with OFCs, previous studies on de novo variants (DNVs) in OFC cases have focused on coding variants that could have a functional impact on protein structure, and the contribution of noncoding DNVs to the formation of OFCs has largely been ignored and is not well understood. MATERIALS AND METHODS:We reanalyzed an existing dataset of DNVs from 1409 trios with OFCs that had undergone targeted sequencing of known OFC-associated loci. We then annotated these DNVs with information from datasets of predicted epigenetic function during human craniofacial development. RESULTS:Of the 66 DNVs called in the targeted regions in this study, 17 (25.7%) were within a predicted enhancer or promoter region. Two DNVs fell within the same enhancer region (hs1617), which is more than expected by chance (p = 0.0017). The sequence changes caused by these hs1617 DNVs are predicted to create binding sites not seen in the reference sequence for transcription factors PAX6 and ZBTB7A and to disrupt binding sites for STAT1 and STAT3. DISCUSSION:The hs1617 enhancer region is within the same topologically associated domain as HHAT, SERTAD4, and IRF6, all of which are involved in craniofacial development. All three genes are highly expressed in human neural crest cells. Knockout mice for Hhat and Irf6 have abnormal embryonic development including a cleft palate, and variants in and around IRF6 are associated with nonsyndromic and syndromic forms of OFCs in humans. CONCLUSION:Taken together, this suggests that noncoding DNVs contribute to the genetic architecture of OFCs, with an excess of DNVs in OFC trios in enhancer regions near known OFC-associated genes. Overall, this adds to our understanding of the genetic mechanisms that underlie OFC formation.

Family-based studies provide a unique opportunity to characterize genetic risks of diseases in the presence of population structure, assortative mating, and indirect genetic effects. We propose a novel framework, PGS-TRI, for the analysis of polygenic scores (PGS) in case-parent trio studies to estimate the risk of an index condition associated with direct effects of inherited PGS, indirect effects of parental PGS, and gene-environment interactions. Extensive simulation studies demonstrate the robustness of PGS-TRI in the presence of complex population structure and assortative mating. We applied PGS-TRI to multi-ancestry trio studies of autism spectrum disorders (ASD) ( N trio = 18 , 383 ), deriving transmission-based estimates of risk for the direct effects of established PGS for ASD and other neurocognitive traits PGS across different ancestry groups and along a genetic ancestry continuum. Our analysis also identified significant indirect effects from parental PGS for BMI and several neurocognitive traits on children's ASD risk. We also employed PGS-TRI in a trio study of European and Asian orofacial clefts (OFCs) ( N trio = 1 , 904 ), investigating both direct and indirect effect of an established PGS and its interaction with maternal risk factors. Finally, we applied PGS-TRI to investigate the direct and indirect effects of large-scale transcriptome-wide and metabolome-wide traits on ASD and OFCs risks.

Nonsyndromic orofacial clefts (OFCs) are common, heritable birth defects caused by both genetic and environmental risk factors. Despite the identification of many genetic loci harboring OFC-risk variants, there are many unknown genetic determinants of OFC. Furthermore, while the process of embryonic facial development is well characterized, the molecular mechanisms that underly it are not. This represents a major hurdle in understanding how disruptions in these biological processes result in OFC. Thus, we sought to identify novel OFC-risk loci through a genome-wide multi-ancestry study of five nested OFC phenotypes (isolated cleft lip [CLO], isolated cleft palate [CPO], cleft lip and palate [CLP], cleft lip with/without cleft palate [CL/P], and any cleft [ANY]) representing distinct cleft subtypes to identify subtype-specific signals and grouped types to maximize power to detect shared genetic effects. We performed genome-wide meta-analyses of these five OFC phenotypes from three cohorts totaling >14,000 individuals using METAL. In addition to replicating 13 known OFC-risk loci, we observed novel association in three regions: the 1p36.32 locus (lead variant rs584402, an intergenic variant, pCLO = 3.14e-8), the 7q33 locus (lead variant rs17168118, an intronic variant in CALD1, pCLP = 9.17e-9), and the 16p13.3 locus (lead variant rs77075754, an intronic variant in RBFOX1, pCL/P = 1.53e-9, pANY = 1.93e-9). We also observed a novel association within the known risk locus 8q22.1 that was independent of the previously reported signal (lead variant rs4735314, an intronic variant in ESRP1, pCLP = 1.07e-9, pCL/P = 3.88e-8). Next, we performed multi-tissue TWAS with s-MulTiXcan and identified four overlapping genes with significant genetically predicted transcription associated with OFC risk. These genes also overlapped the genome-wide significant association signals from the meta-analysis, including CALD1 and ESRP1 and known OFC-risk genes TANC2 and NTN1. Each of the newly reported loci has potential regulatory effects, including evidence of craniofacial enhancer activity, that offer new clues as to the molecule mechanisms underlying embryonic facial development.

De novo variants (DNs) are sporadically occurring variants found in an offspring but absent in both parents. DNs most commonly arise in the germline and are not under selective pressure; therefore, they may be enriched for disease-causing alleles. In fact, DNs have been implicated in multiple rare genetic disorders. Cleft palate (CP) is a craniofacial congenital anomaly occurring in ∼1 in 1,700 live births. Genome-wide association studies have found fewer than a dozen CP-specific loci, while exome and targeted sequencing studies in family-based and case-control cohorts often lack statistical power to conclusively identify causal variants. We therefore hypothesized that CP probands would be enriched for protein-altering DNs, which may explain the relative dearth in discovery. A complicating factor in understanding CP, however, is its phenotypically heterogeneous nature. As such, we aggregated sequence data for 818 trios with CP representing a combination of subtypes and isolated and syndromic presentations. We identified global enrichment of protein-altering DNs (1.48, p = 1.28 × 10-28) and exome-wide-significant (p < 1.3 × 10-6) gene-specific enrichment for SATB2, MEIS2, COL2A1, ZC4H2, EFTUD2, KAT6B, and ANKRD11. We found a statistically significant higher enrichment of protein-altering DNs in syndromic (1.70, p = 6.95 × 10-26) versus nonsyndromic (1.31, p = 8.51 × 10-8) probands but no differences between subtypes. We explored differences in gene-specific enrichment, finding some unique to syndromic probands (ZC4H2) or nonsyndromic probands (IRF6), as well as some shared between groups (SATB2). Altogether, we show that DNs are a contributor to CP risk and that combined analysis can enhance our ability to find genetic associations that would otherwise be undetected.

De novo variants (DNs) are sporadically occurring variants that most commonly arise in the germline and are present in offspring but absent in both parents. As they are not under selective pressure, they may be enriched for disease-causing alleles and have been implicated in multiple rare genetic disorders. Cleft palate (CP) is a common craniofacial congenital anomaly occurring in ~1 in 1700 live births. Genome-wide association studies for CP have found fewer than a dozen loci, while exome and targeted sequencing studies in family-based and case-control cohorts often lack statistical power to conclusively identify causal genes. Based on previous work by our group and others, deciphering the genetic architecture of CP and gene discovery efforts are complicated by the heterogeneous nature of the disorder. We aggregated sequence data for 816 case-parent trios with CP, representing all subtypes of CP and roughly evenly split between isolated and syndromic presentations. We hypothesized there would be a burden of DNs in CP probands and tested this hypothesis in the full cohort and various phenotypic subgroupings. We identified global enrichment of protein-altering DNs (1.36, p=2.39×10-22), and exome-wide significant (p<1.3×10-6) gene-specific enrichment for SATB2, MEIS2, COL2A1, ZC4H2, EFTUD2, KAT6B, and ANKRD11. We found a statistically significant higher enrichment of loss-of-function and missense DNs in syndromic (1.49, p=2.84×10-19) versus nonsyndromic probands (1.25, p=4.01×10-7) but no differences between CP subtypes. We also evaluated biological differences, identifying distinct enrichments across two single cell RNA sequencing datasets: mouse palate at the time of palate fusion and human embryos at post-conceptional weeks 3-5. Altogether, we show DNs are a contributor to CP risk, and that combined analysis can enhance our ability to find genetic associations that would otherwise be undetected.

Objective The objective of this study is to investigate the gene-gene interactions associated with NSCL/P among DNA repair genes. Design This study included 806 NSCL/P case-parent trios from China. Quality control process was conducted for genotyped single nucleotide polymorphisms (SNPs) located in six DNA repair genes ( ATR, ERCC4, RFC1, TYMS, XRCC1 and XRCC3). We tested gene-gene interactions with Cordell's method using statistical package TRIO in R software. Bonferroni corrected significance level was set as P = 4.24 × 10 –4 . We also test the robustness of the interactions by permutation tests. Setting Not applicable. Patients/Participants A total of 806 NSCL/P case-parent trios (complete trios: 682, incomplete trios: 124) with Chinese ancestry. Interventions Not applicable. Main Outcome Measure(s) Not applicable. Results A total of 118 SNPs were extracted for the interaction tests. Fourteen pairs of significant interactions were identified after Bonferroni correction, which were confirmed in permutation tests. Twelve pairs were between ATR and ERCC4 or XRCC3. The most significant interaction occurred between rs2244500 in TYMS and rs3213403 in XRCC1( P = 8.16 × 10 –15 ). Conclusions The current study identified gene-gene interactions among DNA repair genes in 806 Chinese NSCL/P trios, providing additional evidence for the complicated genetic structure underlying NSCL/P. ATR, ERCC4, XRCC3, TYMS and RFC1 were suggested to be possible candidate genes for NSCL/P.

Structural birth defects affect 3-4% of all live births and, depending on the type, tend to manifest in a sex-biased manner. Orofacial clefts (OFCs) are the most common craniofacial structural birth defects and are often divided into cleft lip with or without cleft palate (CL/P) and cleft palate only (CP). Previous studies have found sex-specific risks for CL/P, but these risks have yet to be evaluated in CP. CL/P is more common in males and CP is more frequently observed in females, so we hypothesized there would also be sex-specific differences for CP. Using a trio-based cohort, we performed sex-stratified genome-wide association studies (GWAS) based on proband sex followed by a genome-wide gene-by-sex (GxS) interaction testing. There were 13 loci significant for GxS interactions, with the top finding in LTBP1 (RR=3.37 [2.04 - 5.56], p=1.93x10-6). LTBP1 plays a role in regulating TGF-B bioavailability, and knockdown in both mice and zebrafish lead to craniofacial anomalies. Further, there is evidence for differential expression of LTBP1 between males and females in both mice and humans. Therefore, we tested the association between the imputed genetically regulated gene expression of genes with significant GxS interactions and the CP phenotype. We found significant association for LTBP1 in cell cultured fibroblasts in female probands (p=0.0013) but not in males. Taken altogether, we show there are sex-specific risks for CP that are otherwise undetectable in a combined sex cohort, and LTBP1 is a candidate risk gene, particularly in females.

Novel or rare damaging mutations have been implicated in the developmental pathogenesis of nonsyndromic cleft lip with or without cleft palate (nsCL ± P). Thus, we investigated the human genome for high-impact mutations that could explain the risk of nsCL ± P in our cohorts. We conducted next-generation sequencing (NGS) analysis of 130 nsCL ± P case-parent African trios to identify pathogenic variants that contribute to the risk of clefting. We replicated this analysis using whole-exome sequence data from a Brazilian nsCL ± P cohort. Computational analyses were then used to predict the mechanism by which these variants could result in increased risks for nsCL ± P. We discovered damaging mutations within the AFDN gene, a cell adhesion molecule (CAMs) that was previously shown to contribute to cleft palate in mice. These mutations include p.Met1164Ile, p.Thr453Asn, p.Pro1638Ala, p.Arg669Gln, p.Ala1717Val, and p.Arg1596His. We also discovered a novel splicing p.Leu1588Leu mutation in this protein. Computational analysis suggests that these amino acid changes affect the interactions with other cleft-associated genes including nectins (PVRL1, PVRL2, PVRL3, and PVRL4) CDH1, CTNNA1, and CTNND1. This is the first report on the contribution of AFDN to the risk for nsCL ± P in humans. AFDN encodes AFADIN, an important CAM that forms calcium-independent complexes with nectins 1 and 4 (encoded by the genes PVRL1 and PVRL4). This discovery shows the power of NGS analysis of multiethnic cleft samples in combination with a computational approach in the understanding of the pathogenesis of nsCL ± P.

Abstract Asthma has striking disparities across ancestral groups, but the molecular underpinning of these differences is poorly understood and minimally studied. A goal of the Consortium on Asthma among African-ancestry Populations in the Americas (CAAPA) is to understand multi-omic signatures of asthma focusing on populations of African ancestry. RNASeq and DNA methylation data are generated from nasal epithelium including cases (current asthma, N = 253) and controls (never-asthma, N = 283) from 7 different geographic sites to identify differentially expressed genes (DEGs) and gene networks. We identify 389 DEGs; the top DEG, FN1, was downregulated in cases (q = 3.26 × 10−9) and encodes fibronectin which plays a role in wound healing. The top three gene expression modules implicate networks related to immune response (CEACAM5; p = 9.62 × 10−16 and CPA3; p = 2.39 × 10−14) and wound healing (FN1; p = 7.63 × 10−9). Multi-omic analysis identifies FKBP5, a co-chaperone of glucocorticoid receptor signaling known to be involved in drug response in asthma, where the association between nasal epithelium gene expression is likely regulated by methylation and is associated with increased use of inhaled corticosteroids. This work reveals molecular dysregulation on three axes – increased Th2 inflammation, decreased capacity for wound healing, and impaired drug response – that may play a critical role in asthma within the African Diaspora.

Rationale: The identification of early chronic obstructive pulmonary disease (COPD) is essential to appropriately counsel patients regarding smoking cessation, provide symptomatic treatment, and eventually develop disease-modifying treatments. Disease severity in COPD is defined using race-specific spirometry equations. These may disadvantage non-White individuals in diagnosis and care. Objectives: Determine the impact of race-specific equations on African American (AA) versus non-Hispanic White individuals. Methods: Cross-sectional analyses of the COPDGene (Genetic Epidemiology of Chronic Obstructive Pulmonary Disease) cohort were conducted, comparing non-Hispanic White (n = 6,766) and AA (n = 3,366) participants for COPD manifestations. Measurements and Main Results: Spirometric classifications using race-specific, multiethnic, and "race-reversed" prediction equations (NHANES [National Health and Nutrition Examination Survey] and Global Lung Function Initiative "Other" and "Global") were compared, as were respiratory symptoms, 6-minute-walk distance, computed tomography imaging, respiratory exacerbations, and St. George's Respiratory Questionnaire. Application of different prediction equations to the cohort resulted in different classifications by stage, with NHANES and Global Lung Function Initiative race-specific equations being minimally different, but race-reversed equations moving AA participants to more severe stages and especially between the Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage 0 and preserved ratio impaired spirometry groups. Classification using the established NHANES race-specific equations demonstrated that for each of GOLD stages 1-4, AA participants were younger, had fewer pack-years and more current smoking, but had more exacerbations, shorter 6-minute-walk distance, greater dyspnea, and worse BODE (body mass index, airway obstruction, dyspnea, and exercise capacity) scores and St. George's Respiratory Questionnaire scores. Differences were greatest in GOLD stages 1 and 2. Race-reversed equations reclassified 774 AA participants (43%) from GOLD stage 0 to preserved ratio impaired spirometry. Conclusions: Race-specific equations underestimated disease severity among AA participants. These effects were particularly evident in early disease and may result in late detection of COPD.

RATIONALEIdentification of early COPD is essential to appropriately counsel patients regarding smoking cessation, provide symptomatic treatment and eventually develop disease-modifying treatments. Disease severity in COPD is defined with race-specific spirometry equations. These may disadvantage non-white individuals in diagnosis and care.OBJECTIVESDetermine impact of race-specific equations on African-American (AA) versus non-Hispanic White (NHW) individuals.METHODSCross-sectional analyses of the COPDGene cohort comparing NHW (N=6766) and AA (N=3366) participants for COPD manifestations.MEASUREMENTSSpirometric classifications using race-specific, multi-ethnic and race-reversed prediction equations [National Health and Nutrition Examination Survey (NHANES) and Global Lung Function Initiative (GLI) "Other and Global"] were compared, as were respiratory symptoms, six-minute walk distance (6MWD), CT imaging, respiratory exacerbations, and St. George's Respiratory Questionnaire (SGRQ).MAIN RESULTSApplication of different prediction equations to the cohort resulted in different classifications by stage, with NHANES and GLI race-specific equations being minimally different, but "race-reversed" equations moving AA to more severe stages and especially between the GOLD 0 and preserved ratio impaired spirometry (PRISm) groups. Classification using the established NHANES race-specific equations demonstrated that for each GOLD stage 1-4, AA participants were younger, had fewer pack years and more current smoking, but had more exacerbations, lower 6MWD, greater dyspnea and worse BODE scores and SGRQ scores. Differences were greatest in GOLD 1 and 2 stages. Race-reversed equations reclassified 774 AA (43%) from GOLD 0 to PRISm.CONCLUSIONSRace-specific equations underestimate disease severity among AAs. These effects are particularly evident in early disease and may result in late detection of COPD.

Abstract Background The association between genetic variants on the X chromosome to risk of COPD has not been fully explored. We hypothesize that the X chromosome harbors variants important in determining risk of COPD related phenotypes and may drive sex differences in COPD manifestations. Methods Using X chromosome data from three COPD-enriched cohorts of adult smokers, we performed X chromosome specific quality control, imputation, and testing for association with COPD case–control status, lung function, and quantitative emphysema. Analyses were performed among all subjects, then stratified by sex, and subsequently combined in meta-analyses. Results Among 10,193 subjects of non-Hispanic white or European ancestry, a variant near TMSB4X, rs5979771, reached genome-wide significance for association with lung function measured by FEV1/FVC ( $$\beta$$ β 0.020, SE 0.004, p 4.97 × 10–08), with suggestive evidence of association with FEV1 ( $$\beta$$ β 0.092, SE 0.018, p 3.40 × 10–07). Sex-stratified analyses revealed X chromosome variants that were differentially trending in one sex, with significantly different effect sizes or directions. Conclusions This investigation identified loci influencing lung function, COPD, and emphysema in a comprehensive genetic association meta-analysis of X chromosome genetic markers from multiple COPD-related datasets. Sex differences play an important role in the pathobiology of complex lung disease, including X chromosome variants that demonstrate differential effects by sex and variants that may be relevant through escape from X chromosome inactivation. Comprehensive interrogation of the X chromosome to better understand genetic control of COPD and lung function is important to further understanding of disease pathology. Trial registration Genetic Epidemiology of COPD Study (COPDGene) is registered at ClinicalTrials.gov, NCT00608764 (Active since January 28, 2008). Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints Study (ECLIPSE), GlaxoSmithKline study code SCO104960, is registered at ClinicalTrials.gov, NCT00292552 (Active since February 16, 2006). Genetics of COPD in Norway Study (GenKOLS) holds GlaxoSmithKline study code RES11080, Genetics of Chronic Obstructive Lung Disease.

Additional file 4: Table S3. Replication Examination Of Associations From Other Studies In This XWAS Meta-analysis

Rationale: Cigarette smoking contributes to risk of death through different mechanisms. Objectives: To determine how causes of and clinical features associated with death vary in tobacco cigarette users by lung function impairment. Methods: We stratified current and former tobacco cigarette users enrolled in COPDGene into normal spirometry, Preserved Ratio Impaired Spirometry (PRISm), GOLD 1-2 and GOLD 3-4 COPD. Deaths were identified via longitudinal follow-up and Social Security Death Index search. Causes of death were adjudicated after review of death certificates, medical records and next-of-kin interviews. We tested associations between baseline clinical variables and all-cause mortality using multivariable Cox proportional-hazards models. Results: Over 10.1-year median follow-up, 2,200 deaths occurred among 10,132 participants (age 59.5±9.0 years; 46.6% women). Death from cardiovascular disease was most frequent in PRISm (31% of deaths). Lung cancer deaths were most frequent in GOLD 1-2 (18% of deaths vs 9-11% in other groups). Respiratory deaths outpaced competing causes of death in GOLD 3-4, particularly when BODE index ≥7. St. George's Respiratory Questionnaire score ≥25 was associated with higher mortality in all groups: HR 1.48 [1.20-1.84] normal spirometry; HR 1.40 [1.05-1.87] PRISm; HR 1.80 [1.49-2.17] GOLD 1-2; HR 1.65 [1.26-2.17] GOLD 3-4. History of respiratory exacerbations was associated with higher mortality in GOLD 1-2 and GOLD 3-4, quantitative emphysema in GOLD 1-2, and airway wall thickness in PRISm and GOLD 3-4. Conclusions: Leading causes of death vary by lung function impairment in tobacco cigarette users. Worse respiratory-related quality of life is associated with all-cause mortality regardless of lung function.

Additional file 3: Table S2. XWAS Meta-analysis Top ACE2 Xp22.2 Variants