Pulmonary Function and Blood DNA Methylation: A Multiancestry Epigenome-Wide Association Meta-analysis.

Rationale: Methylation integrates factors present at birth and modifiable across the lifespan that can influence pulmonary function. Studies are limited in scope and replication. Objectives: To conduct large-scale epigenome-wide meta-analyses of blood DNA methylation and pulmonary function. Methods: Twelve cohorts analyzed associations of methylation at cytosine-phosphate-guanine probes (CpGs), using Illumina 450K or EPIC/850K arrays, with FEV1, FVC, and FEV1/FVC. We performed multiancestry epigenome-wide meta-analyses (total of 17,503 individuals; 14,761 European, 2,549 African, and 193 Hispanic/Latino ancestries) and interpreted results using integrative epigenomics. Measurements and Main Results: We identified 1,267 CpGs (1,042 genes) differentially methylated (false discovery rate, <0.025) in relation to FEV1, FVC, or FEV1/FVC, including 1,240 novel and 73 also related to chronic obstructive pulmonary disease (1,787 cases). We found 294 CpGs unique to European or African ancestry and 395 CpGs unique to never or ever smokers. The majority of significant CpGs correlated with nearby gene expression in blood. Findings were enriched in key regulatory elements for gene function, including accessible chromatin elements, in both blood and lung. Sixty-nine implicated genes are targets of investigational or approved drugs. One example novel gene highlighted by integrative epigenomic and druggable target analysis is TNFRSF4. Mendelian randomization and colocalization analyses suggest that epigenome-wide association study signals capture causal regulatory genomic loci. Conclusions: We identified numerous novel loci differentially methylated in relation to pulmonary function; few were detected in large genome-wide association studies. Integrative analyses highlight functional relevance and potential therapeutic targets. This comprehensive discovery of potentially modifiable, novel lung function loci expands knowledge gained from genetic studies, providing insights into lung pathogenesis.

A Pilot Study Examining the Effects of Ischemic Conditioning on Walking Capacity and Lower Extremity Muscle Performance in Patients with Claudication.

INTRODUCTION: This study investigated whether a novel therapy called ischemic conditioning (IC) improves walking capacity and lower extremity muscle performance in patients with peripheral vascular disease who experience intermittent claudication. METHODS: Forty-three patients with claudication were enrolled and received either IC or IC Sham for 2 weeks in this randomized, controlled, double-blinded, prospective study. IC sessions involved five cycles of alternating 5-min inflations of a blood pressure cuff to 225 mm Hg (25 mm Hg for IC Sham) and 5-min deflations, around the thigh of the affected lower extremity. RESULTS: There was no difference in the change in claudication onset time (Δ = 114 ± 212 s IC vs. 104 ± 173 s IC Sham; p = 0.67) or peak walking time (Δ = 42 ± 139 s IC vs. 12 ± 148 s IC Sham; p = 0.35) between the IC and IC Sham groups. At the level of the knee, participants in the IC group performed more work (Δ = 3,029 ± 4,999 J IC vs. 345 ± 2,863 J IC Sham; p = 0.03) and displayed a greater time to muscle fatigue (Δ = 147 ± 221 s IC vs. -27 ± 236 s IC Sham; p = 0.01). DISCUSSION/CONCLUSION: In patients with claudication, IC improved total work performed and time to fatigue at the knee but did not change walking parameters.

A systematic analysis of protein-altering exonic variants in chronic obstructive pulmonary disease.

Genome-wide association studies (GWASs) have identified regions associated with chronic obstructive pulmonary disease (COPD). GWASs of other diseases have shown an approximately 10-fold overrepresentation of nonsynonymous variants, despite limited exonic coverage on genotyping arrays. We hypothesized that a large-scale analysis of coding variants could discover novel genetic associations with COPD, including rare variants with large effect sizes. We performed a meta-analysis of exome arrays from 218,399 controls and 33,851 moderate-to-severe COPD cases. All exome-wide significant associations were present in regions previously identified by GWAS. We did not identify any novel rare coding variants with large effect sizes. Within GWAS regions on chromosomes 5q, 6p, and 15q, four coding variants were conditionally significant (P < 0.00015) when adjusting for lead GWAS single-nucleotide polymorphisms A common gasdermin B (GSDMB) splice variant (rs11078928) previously associated with a decreased risk for asthma was nominally associated with a decreased risk for COPD [minor allele frequency (MAF) = 0.46, P = 1.8e-4]. Two stop variants in coiled-coil α-helical rod protein 1 (CCHCR1), a gene involved in regulating cell proliferation, were associated with COPD (both P < 0.0001). The SERPINA1 Z allele was associated with a random-effects odds ratio of 1.43 for COPD (95% confidence interval = 1.17-1.74), though with marked heterogeneity across studies. Overall, COPD-associated exonic variants were identified in genes involved in DNA methylation, cell-matrix interactions, cell proliferation, and cell death. In conclusion, we performed the largest exome array meta-analysis of COPD to date and identified potential functional coding variants. Future studies are needed to identify rarer variants and further define the role of coding variants in COPD pathogenesis.

Human airway branch variation and chronic obstructive pulmonary disease.

Susceptibility to chronic obstructive pulmonary disease (COPD) beyond cigarette smoking is incompletely understood, although several genetic variants associated with COPD are known to regulate airway branch development. We demonstrate that in vivo central airway branch variants are present in 26.5% of the general population, are unchanged over 10 y, and exhibit strong familial aggregation. The most common airway branch variant is associated with COPD in two cohorts (n = 5,054), with greater central airway bifurcation density, and with emphysema throughout the lung. The second most common airway branch variant is associated with COPD among smokers, with narrower airway lumens in all lobes, and with genetic polymorphisms within the FGF10 gene. We conclude that central airway branch variation, readily detected by computed tomography, is a biomarker of widely altered lung structure with a genetic basis and represents a COPD susceptibility factor.

A Genetic Risk Score Associated with Chronic Obstructive Pulmonary Disease Susceptibility and Lung Structure on Computed Tomography.

Rationale: Chronic obstructive pulmonary disease (COPD) has been associated with numerous genetic variants, yet the extent to which its genetic risk is mediated by variation in lung structure remains unknown.Objectives: To characterize associations between a genetic risk score (GRS) associated with COPD susceptibility and lung structure on computed tomography (CT).Methods: We analyzed data from MESA Lung (Multi-Ethnic Study of Atherosclerosis Lung Study), a U.S. general population-based cohort, and SPIROMICS (Subpopulations and Intermediate Outcome Measures in COPD Study). A weighted GRS was calculated from 83 SNPs that were previously associated with lung function. Lung density, spatially matched airway dimensions, and airway counts were assessed on full-lung CT. Generalized linear models were adjusted for age, age squared, sex, height, principal components of genetic ancestry, smoking status, pack-years, CT model, milliamperes, and total lung volume.Measurements and Main Results: MESA Lung and SPIROMICS contributed 2,517 and 2,339 participants, respectively. Higher GRS was associated with lower lung function and increased COPD risk, as well as lower lung density, smaller airway lumens, and fewer small airways, without effect modification by smoking. Adjustment for CT lung structure, particularly small airway measures, attenuated associations between the GRS and FEV1/FVC by 100% and 60% in MESA and SPIROMICS, respectively. Lung structure (P < 0.0001), but not the GRS (P > 0.10), improved discrimination of moderate-to-severe COPD cases relative to clinical factors alone.Conclusions: A GRS associated with COPD susceptibility was associated with CT lung structure. Lung structure may be an important mediator of heritability and determinant of personalized COPD risk.

Genome-wide association study of subclinical interstitial lung disease in MESA.

BACKGROUND: We conducted a genome-wide association study (GWAS) of subclinical interstitial lung disease (ILD), defined as high attenuation areas (HAA) on CT, in the population-based Multi-Ethnic Study of Atherosclerosis Study. METHODS: We measured the percentage of high attenuation areas (HAA) in the lung fields on cardiac CT scan defined as voxels with CT attenuation values between -600 and -250 HU. Genetic analyses were performed in MESA combined across race/ethnic groups: non-Hispanic White (n = 2,434), African American (n = 2,470), Hispanic (n = 2,065) and Chinese (n = 702), as well as stratified by race/ethnicity. RESULTS: Among 7,671 participants, regions at genome-wide significance were identified for basilar peel-core ratio of HAA in FLJ35282 downstream of ANRIL (rs7852363, P = 2.1x10-9) and within introns of SNAI3-AS1 (rs140142658, P = 9.6x10-9) and D21S2088E (rs3079677, P = 2.3x10-8). Within race/ethnic groups, 18 additional loci were identified at genome-wide significance, including genes related to development (FOXP4), cell adhesion (ALCAM) and glycosylation (GNPDA2, GYPC, GFPT1 and FUT10). Among these loci, SNP rs6844387 near GNPDA2 demonstrated nominal evidence of replication in analysis of n = 1,959 participants from the Framingham Heart Study (P = 0.029). FOXP4 region SNP rs2894439 demonstrated evidence of validation in analysis of n = 228 White ILD cases from the Columbia ILD Study compared to race/ethnicity-matched controls from MESA (one-sided P = 0.007). In lung tissue from 15 adults with idiopathic pulmonary fibrosis compared to 15 adults without lung disease. ANRIL (P = 0.001), ALCAM (P = 0.03) and FOXP4 (P = 0.046) were differentially expressed. CONCLUSIONS: Our results suggest novel roles for protein glycosylation and cell cycle disinhibition by long non-coding RNA in the pathogenesis of ILD.

Non-Invasive Estimation of Skeletal Muscle Oxygen Consumption Rate and Microvascular Reactivity in Chronic Stroke Survivors using Near Infrared Spectroscopy.

BACKGROUND: Understanding post-stroke changes in skeletal muscle oxidative metabolism and microvascular reactivity could help create therapeutic targets that optimize rehabilitative interventions. Due to disuse atrophy, we hypothesized that basal muscle oxygen consumption rate and microvascular endothelial function would be impaired in the tibialis anterior (TA) muscle of the affected leg of chronic stroke survivors compared to the non-affected leg and vs. matched controls. METHODS: Fifteen chronic stroke survivors (10 female) and 15 matched controls (9 female) completed this study. A near infrared spectroscopy oximeter measured tissue oxygen saturation (StO2) of the TA in both legs of stroke survivors and the dominant leg of controls. A cuff was placed around the thigh and inflated to 225 mmHg for five minutes while StO2 was continuously measured. The rate of change in StO2 was calculated during cuff occlusion and immediately post-cuff release. RESULTS: The rate of oxygen desaturation was similar between the legs of the stroke survivors (paretic -0.12±0.04 %∙s-1 vs non-paretic -0.16±011 %∙s-1; p=0.49), but the paretic leg had a reduced desaturation rate vs. controls (-0.25±0.18%∙s-1; p=0.007 vs. paretic leg). After cuff release, there was a greater oxygen resaturation rate in the non-paretic leg compared to the paretic leg (3.13±2.08 %∙s-1 vs. 1.60±1.11 %∙s-1, respectively; p=0.01). The control leg had a similar resaturation rate vs. the non-paretic leg (control = 3.41±1.79%∙s-1; p=0.69) but was greater than the paretic leg (p=0.003). CONCLUSION: The TA in the paretic leg had an impaired muscle oxygen consumption rate and reduced microvascular endothelial function compared to controls.

Reduced oxygen desaturation in the vastus lateralis of chronic stroke survivors during graded muscle contractions.

BACKGROUND: Few studies have examined changes in skeletal muscle physiology post-stroke. This study examined changes in tissue oxygen saturation (StO2) of the vastus lateralis (VL) muscle of stroke survivors and age-matched control participants during maximal and submaximal isometric contractions of the knee extensor muscles. OBJECTIVES: We hypothesized that tissue oxygen desaturation (ΔStO2) during knee extensor muscle contractions would be less in the VL in the paretic vs. the non-paretic and control legs. METHODS: Ten chronic stroke survivors (>6 months post-stroke) with lower extremity muscle weakness and 10 age-matched controls completed this prospective cohort study. Maximum voluntary contractions (MVCs) of the knee extensor muscles were assessed with a Biodex dynamometer and StO2 of the VL was measured using near-infrared spectroscopy. RESULTS: In the paretic leg of the stroke survivors little change in StO2 of the VL was observed during an MVC (ΔStO2 = -1.7 ± 1.8%) compared to the non-paretic (ΔStO2 = -5.1 ± 6.1%; p < 0.05) and control legs (ΔStO2 = -14.4 ± 8.8%; p < 0.05 vs. paretic and non-paretic leg). These differences remained when normalizing for strength differences between the legs. Compared to controls, both the paretic and non-paretic VL showed pronounced reductions in ΔStO2 during ramp and hold contractions equal to 20%, 40%, or 60% of the MVC (p < 0.05 vs. controls at all load levels). CONCLUSIONS: These results indicate that oxygen desaturation in response to isometric muscle contractions is impaired in both the paretic and non-paretic leg muscle of stroke survivors compared to age-matched controls, and these differences are independent of differences in muscle strength.

Quantification of Tissue Oxygen Saturation in the Vastus Lateralis Muscle of Chronic Stroke Survivors during a Graded Exercise Test.

Purpose: This study examined tissue oxygen saturation (StO2) of the vastus lateralis (VL) muscles of chronic stroke survivors during a graded exercise test (GXT). We hypothesized the reduction in StO2 will be blunted in the paretic vs. non-paretic VL during a maximum-effort GXT. Methods: Chronic stroke survivors performed a GXT and StO2 of the VL in each leg was measured using near infrared spectroscopy. Twenty-six stroke survivors performed a GXT. Results: At rest, there was no difference in StO2 between the paretic and non-paretic VL (65±9% vs. 68±7%, respectively, p=0.32). The maximum change in StO2 from rest during the GXT was greater in the non-paretic vs. the paretic VL (-16±14% vs. -9±10%, respectively, p<0.001). The magnitude of the oxygen resaturation response was also greater in the non-paretic vs. the paretic VL (29±23% vs. 18±15%, respectively, p<0.001). VO2 Peak was associated with the magnitude of the VL StO2 change during (r2=0.54, p<0.0001) and after (r2=0.56, p<0.001) the GXT. Conclusions: During a GXT there is a blunted oxygen desaturation response in the paretic vs. the non-paretic VL of chronic stroke survivors. In the paretic VL there was a positive correlation between the oxygen desaturation response during the GXT and VO2 Peak.

Impaired Firing Behavior of Individually Tracked Paretic Motor Units During Fatiguing Contractions of the Dorsiflexors and Functional Implications Post Stroke.

Introduction: This study quantified stroke-related changes in the following: (1) the averaged discharge rate of motor units (individually tracked and untracked) identified from high-density electromyography (HD-EMG) recordings, (2) global muscle EMG properties of the dorsiflexors during a fatiguing contraction, and the relationship between task endurance and measures of leg function. Methods: Ten individuals with chronic stroke performed a sustained sub-maximal, isometric, fatiguing dorsiflexion contraction in paretic and non-paretic legs. Motor-unit firing behavior, task duration, maximal voluntary contraction strength (MVC), and clinical measures of leg function were obtained. Results: Compared to the non-paretic leg, the paretic leg task duration was shorter, and there was a larger exercise-related reduction in motor unit global rates, individually tracked discharge rates, and overall magnitude of EMG. Task duration of the paretic leg was more predictive of walking speed and lower extremity Fugl-Meyer scores compared to the non-paretic leg. Discussion: Paretic leg muscle fatigability is increased post stroke. It is characterized by impaired rate coding and recruitment and relates to measures of motor function.

Two weeks of remote ischemic conditioning improves brachial artery flow mediated dilation in chronic stroke survivors.

Many stroke survivors have reduced cardiorespiratory fitness as a result of their stroke. Ischemic conditioning (IC) is a noninvasive, cost-effective, easy-to-administer intervention that can be performed at home and has been shown to improve both motor function in stroke survivors and vascular endothelial function in healthy individuals. In this study, we examined the effects of 2 wk of remote IC (RIC) on brachial artery flow mediated dilation (FMD) in chronic stroke survivors. We hypothesized that FMD would be improved following RIC compared with a sham RIC control group. This was a prospective, randomized, double-blinded, controlled study. Twenty-four chronic stroke survivors (>6 mo after stroke) were enrolled and randomized to receive either RIC or sham RIC on their affected thigh every other day for 2 wk. For the RIC group, a blood pressure cuff was inflated to 225 mmHg for 5 min, followed by 5 min of recovery, and repeated a total of five times per session. For the sham RIC group, the inflation pressure was 10 mmHg. Brachial artery FMD was assessed on the nonaffected arm at study enrollment and following the 2-wk intervention period. Nine men and fourteen women completed all study procedures. Brachial artery FMD increased from 5.4 ± 4.8 to 7.8 ± 4.4% (P = 0.030; n = 12) in the RIC group, while no significant change was observed in the sham RIC group (3.5 ± 3.9% pretreatment versus 2.4 ± 3.1% posttreatment; P = 0.281, n = 11). Two weeks of RIC increases brachial artery FMD in chronic stroke survivors.NEW & NOTEWORTHY In this study, we report that 2 wk of remote ischemic conditioning (RIC) improves brachial artery flow-mediated dilation in chronic stroke survivors. Because poor cardiovascular health puts stroke survivors at a heightened risk for recurrent stroke and other cardiovascular events, an intervention that is simple, cost-effective, and easy to perform like RIC holds promise as a means to improve cardiovascular health in this at-risk population.

Two weeks of ischemic conditioning improves walking speed and reduces neuromuscular fatigability in chronic stroke survivors.

This pilot study examined whether ischemic conditioning (IC), a noninvasive, cost-effective, and easy-to-administer intervention, could improve gait speed and paretic leg muscle function in stroke survivors. We hypothesized that 2 wk of IC training would increase self-selected walking speed, increase paretic muscle strength, and reduce neuromuscular fatigability in chronic stroke survivors. Twenty-two chronic stroke survivors received either IC or IC Sham on their paretic leg every other day for 2 wk (7 total sessions). IC involved 5-min bouts of ischemia, repeated five times, using a cuff inflated to 225 mmHg on the paretic thigh. For IC Sham, the cuff inflation pressure was 10 mmHg. Self-selected walking speed was assessed using the 10-m walk test, and paretic leg knee extensor strength and fatigability were assessed using a Biodex dynamometer. Self-selected walking speed increased in the IC group (0.86 ± 0.21 m/s pretest vs. 1.04 ± 0.22 m/s posttest, means ± SD; P < 0.001) but not in the IC Sham group (0.92 ± 0.47 m/s pretest vs. 0.96 ± 0.46 m/s posttest; P = 0.25). Paretic leg maximum voluntary contractions were unchanged in both groups (103 ± 57 N·m pre-IC vs. 109 ± 65 N·m post-IC; 103 ± 59 N·m pre-IC Sham vs. 108 ± 67 N·m post-IC Sham; P = 0.81); however, participants in the IC group maintained a submaximal isometric contraction longer than participants in the IC Sham group (278 ± 163 s pre-IC vs. 496 ± 313 s post-IC, P = 0.004; 397 ± 203 s pre-IC Sham vs. 355 ± 195 s post-IC Sham; P = 0.46). The results from this pilot study thus indicate that IC training has the potential to improve walking speed and paretic muscle fatigue resistance poststroke. NEW & NOTEWORTHY This pilot study is the first to demonstrate that ischemic conditioning can improve self-selected walking speed and reduce paretic muscle fatigue in stroke survivors. Ischemic conditioning has been shown to be safe in numerous patient populations, can be accomplished at home or at the bedside in only 45 min, and requires no specialized training. Future larger studies are warranted to determine the efficacy of ischemic conditioning as a neurorehabilitation therapy poststroke.

Prognostic Significance of Large Airway Dimensions on Computed Tomography in the General Population. The Multi-Ethnic Study of Atherosclerosis (MESA) Lung Study.

RATIONALE: Large airway dimensions on computed tomography (CT) have been associated with lung function, symptoms, and exacerbations in chronic obstructive pulmonary disease (COPD), as well as with symptoms in smokers with preserved spirometry. Their prognostic significance in persons without lung disease remains undefined. OBJECTIVES: To examine associations between large airway dimensions on CT and respiratory outcomes in a population-based cohort of adults without prevalent lung disease. METHODS: The Multi-Ethnic Study of Atherosclerosis recruited participants ages 45-84 years without cardiovascular disease in 2000-2002; we excluded participants with prevalent chronic lower respiratory disease (CLRD). Spirometry was measured in 2004-2006 and 2010-2012. CLRD hospitalizations and deaths were classified by validated criteria through 2014. The average wall thickness for a hypothetical airway of 10-mm lumen perimeter on CT (Pi10) was calculated using measures of airway wall thickness and lumen diameter. Models were adjusted for age, sex, principal components of ancestry, body mass index, smoking, pack-years, scanner, percent emphysema, genetic risk score, and initial forced expiratory volume in 1 second (FEV1) percent predicted. RESULTS: Greater Pi10 was associated with 9% faster FEV1 decline (95% confidence interval [CI], 2 to 15%; P = 0.012) and increased incident COPD (odds ratio, 2.22; 95% CI, 1.43-3.45; P = 0.0004) per standard deviation among 1,830 participants. Over 78,147 person-years, higher Pi10 was associated with a 57% higher risk of first CLRD hospitalization or mortality (P = 0.0496) per standard deviation. Of Pi10's component measures, both greater airway wall thickness and narrower lumen predicted incident COPD and CLRD clinical events. CONCLUSIONS: In adults without CLRD, large airway dimensions on CT were prospectively associated with accelerated lung function decline and increased risks of COPD and CLRD hospitalization and mortality.

Multiethnic meta-analysis identifies ancestry-specific and cross-ancestry loci for pulmonary function.

Nearly 100 loci have been identified for pulmonary function, almost exclusively in studies of European ancestry populations. We extend previous research by meta-analyzing genome-wide association studies of 1000 Genomes imputed variants in relation to pulmonary function in a multiethnic population of 90,715 individuals of European (N = 60,552), African (N = 8429), Asian (N = 9959), and Hispanic/Latino (N = 11,775) ethnicities. We identify over 50 additional loci at genome-wide significance in ancestry-specific or multiethnic meta-analyses. Using recent fine-mapping methods incorporating functional annotation, gene expression, and differences in linkage disequilibrium between ethnicities, we further shed light on potential causal variants and genes at known and newly identified loci. Several of the novel genes encode proteins with predicted or established drug targets, including KCNK2 and CDK12. Our study highlights the utility of multiethnic and integrative genomics approaches to extend existing knowledge of the genetics of lung function and clinical relevance of implicated loci.

Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease.

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.