Reduction of Z alpha-1 antitrypsin polymers in human iPSC-hepatocytes and mice by LRRK2 inhibitors.

Alpha-1 antitrypsin deficiency (A1ATD) is a life-threatening condition caused by inheritance of the SERPINA1 'Z' genetic variant (PiZ) driving AAT protein misfolding in hepatocytes. There remain no approved medicines for this disease. Here, we report the results of a small molecule screen performed in patient derived iPSC-hepatocytes that identified Leucine-rich repeat kinase-2 (LRRK2) as a potentially new therapeutic target. Of the commercially available LRRK2 inhibitors tested, we identified CZC-25146, a candidate with favorable pharmacokinetic properties, as being capable of reducing polymer load, increasing normal AAT secretion, and reducing inflammatory cytokines in both cells and PiZ mice. Mechanistically, this effect was achieved through induction of autophagy. Our findings support the use of CZC-25146 and LRRK2 inhibitors in hepatic proteinopathy research and their further investigation as novel therapeutic candidates for A1ATD.

Conformational pathology of the serpins: themes, variations, and therapeutic strategies.

Point mutations cause members of the serine protease inhibitor (serpin) superfamily to undergo a novel conformational transition, forming ordered polymers. These polymers characterize a group of diseases termed the serpinopathies. The formation of polymers underlies the retention of alpha(1)-antitrypsin within hepatocytes and of neuroserpin within neurons to cause cirrhosis and dementia, respectively. Point mutations of antithrombin, C1 inhibitor, alpha(1)-antichymotrypsin, and heparin cofactor II cause a similar conformational transition, resulting in a plasma deficiency that is associated with thrombosis, angioedema, and emphysema. Polymers of serpins can also form in extracellular tissues where they activate inflammatory cascades. This is best described for the Z variant of alpha(1)-antitrypsin in which the proinflammatory properties of polymers provide an explanation for both progressive emphysema and the selective advantage of this mutant allele. Therapeutic strategies are now being developed to block the aberrant conformational transitions and so treat the serpinopathies.

X chromosome associations with chronic obstructive pulmonary disease and related phenotypes: an X chromosome-wide association study.

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 ([Formula: see text] 0.020, SE 0.004, p 4.97 × 10-08), with suggestive evidence of association with FEV1 ([Formula: see text] 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.

Keratin 8 is a scaffolding and regulatory protein of ERAD complexes.

Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild-type (WT) A1AT with a 13-fold and fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the µs heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation and co-immunoprecipitation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients' respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.

Hepatobiliary phenotypes of adults with alpha-1 antitrypsin deficiency.

OBJECTIVE: Alpha-1 antitrypsin deficiency (AATD) is a common, potentially lethal inborn disorder caused by mutations in alpha-1 antitrypsin (AAT). Homozygosity for the 'Pi*Z' variant of AAT (Pi*ZZ genotype) causes lung and liver disease, whereas heterozygous 'Pi*Z' carriage (Pi*MZ genotype) predisposes to gallstones and liver fibrosis. The clinical significance of the more common 'Pi*S' variant remains largely undefined and no robust data exist on the prevalence of liver tumours in AATD. DESIGN: Baseline phenotypes of AATD individuals and non-carriers were analysed in 482 380 participants in the UK Biobank. 1104 participants of a multinational cohort (586 Pi*ZZ, 239 Pi*SZ, 279 non-carriers) underwent a comprehensive clinical assessment. Associations were adjusted for age, sex, body mass index, diabetes and alcohol consumption. RESULTS: Among UK Biobank participants, Pi*ZZ individuals displayed the highest liver enzyme values, the highest occurrence of liver fibrosis/cirrhosis (adjusted OR (aOR)=21.7 (8.8-53.7)) and primary liver cancer (aOR=44.5 (10.8-183.6)). Subjects with Pi*MZ genotype had slightly elevated liver enzymes and moderately increased odds for liver fibrosis/cirrhosis (aOR=1.7 (1.2-2.2)) and cholelithiasis (aOR=1.3 (1.2-1.4)). Individuals with homozygous Pi*S mutation (Pi*SS genotype) harboured minimally elevated alanine aminotransferase values, but no other hepatobiliary abnormalities. Pi*SZ participants displayed higher liver enzymes, more frequent liver fibrosis/cirrhosis (aOR=3.1 (1.1-8.2)) and primary liver cancer (aOR=6.6 (1.6-26.9)). The higher fibrosis burden was confirmed in a multinational cohort. Male sex, age ≥50 years, obesity and the presence of diabetes were associated with significant liver fibrosis. CONCLUSION: Our study defines the hepatobiliary phenotype of individuals with the most relevant AATD genotypes including their predisposition to liver tumours, thereby allowing evidence-based advice and individualised hepatological surveillance.

Once-Daily Single-Inhaler Triple versus Dual Therapy in Patients with COPD.

BACKGROUND: The benefits of triple therapy for chronic obstructive pulmonary disease (COPD) with an inhaled glucocorticoid, a long-acting muscarinic antagonist (LAMA), and a long-acting ß2-agonist (LABA), as compared with dual therapy (either inhaled glucocorticoid-LABA or LAMA-LABA), are uncertain. METHODS: In this randomized trial involving 10,355 patients with COPD, we compared 52 weeks of a once-daily combination of fluticasone furoate (an inhaled glucocorticoid) at a dose of 100 µg, umeclidinium (a LAMA) at a dose of 62.5 µg, and vilanterol (a LABA) at a dose of 25 µg (triple therapy) with fluticasone furoate-vilanterol (at doses of 100 µg and 25 µg, respectively) and umeclidinium-vilanterol (at doses of 62.5 µg and 25 µg, respectively). Each regimen was administered in a single Ellipta inhaler. The primary outcome was the annual rate of moderate or severe COPD exacerbations during treatment. RESULTS: The rate of moderate or severe exacerbations in the triple-therapy group was 0.91 per year, as compared with 1.07 per year in the fluticasone furoate-vilanterol group (rate ratio with triple therapy, 0.85; 95% confidence interval [CI], 0.80 to 0.90; 15% difference; P<0.001) and 1.21 per year in the umeclidinium-vilanterol group (rate ratio with triple therapy, 0.75; 95% CI, 0.70 to 0.81; 25% difference; P<0.001). The annual rate of severe exacerbations resulting in hospitalization in the triple-therapy group was 0.13, as compared with 0.19 in the umeclidinium-vilanterol group (rate ratio, 0.66; 95% CI, 0.56 to 0.78; 34% difference; P<0.001). There was a higher incidence of pneumonia in the inhaled-glucocorticoid groups than in the umeclidinium-vilanterol group, and the risk of clinician-diagnosed pneumonia was significantly higher with triple therapy than with umeclidinium-vilanterol, as assessed in a time-to-first-event analysis (hazard ratio, 1.53; 95% CI, 1.22 to 1.92; P<0.001). CONCLUSIONS: Triple therapy with fluticasone furoate, umeclidinium, and vilanterol resulted in a lower rate of moderate or severe COPD exacerbations than fluticasone furoate-vilanterol or umeclidinium-vilanterol in this population. Triple therapy also resulted in a lower rate of hospitalization due to COPD than umeclidinium-vilanterol. (Funded by GlaxoSmithKline; IMPACT ClinicalTrials.gov number, NCT02164513 .).

Markers of disease activity in COPD: an 8-year mortality study in the ECLIPSE cohort.

RATIONALE: There are no validated measures of disease activity in COPD. Since "active" disease is expected to have worse outcomes (e.g. mortality), we explored potential markers of disease activity in patients enrolled in the ECLIPSE cohort in relation to 8-year all-cause mortality. METHODS: We investigated 1) how changes in relevant clinical variables over time (1 or 3 years) relate to 8-year mortality; 2) whether these variables inter-relate; and 3) if any clinical, imaging and/or biological marker measured cross-sectionally at baseline relates to any activity component. RESULTS: Results showed that 1) after 1 year, hospitalisation for COPD, exacerbation frequency, worsening of body mass index, airflow obstruction, dyspnoea and exercise (BODE) index or health status (St George's Respiratory Questionnaire (SGRQ)) and persistence of systemic inflammation were significantly associated with 8-year mortality; 2) at 3 years, the same markers, plus forced expiratory volume in 1 s (FEV1) decline and to a lesser degree computed tomography (CT) emphysema, showed association, thus qualifying as markers of disease activity; 3) changes in FEV1, inflammatory cytokines and CT emphysema were not inter-related, while the multidimensional indices (BODE and SGRQ) showed modest correlations; and 4) changes in these markers could not be predicted by any baseline cross-sectional measure. CONCLUSIONS: In COPD, 1- and 3-year changes in exacerbation frequency, systemic inflammation, BODE and SGRQ scores and FEV1 decline are independent markers of disease activity associated with 8-year all-cause mortality. These disease activity markers are generally independent and not predictable from baseline measurements.

The development of highly potent and selective small molecule correctors of Z α1-antitrypsin misfolding.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.

The Effect of Inhaled Corticosteroid Withdrawal and Baseline Inhaled Treatment on Exacerbations in the IMPACT Study. A Randomized, Double-Blind, Multicenter Clinical Trial.

Rationale: In the IMPACT (Informing the Pathway of Chronic Obstructive Pulmonary Disease Treatment) trial, fluticasone furoate (FF)/umeclidinium (UMEC)/vilanterol (VI) significantly reduced exacerbations compared with FF/VI or UMEC/VI in patients with symptomatic chronic obstructive pulmonary disease and a history of exacerbations.Objectives: To understand whether inhaled corticosteroid (ICS) withdrawal affected IMPACT results, given direct transition from prior maintenance medication to study medication at randomization.Methods: Exacerbations and change from baseline in trough FEV1 and St. George's Respiratory Questionnaire results were analyzed by prior ICS use. Exacerbations were also analyzed while excluding data from the first 30 days.Measurements and Main Results: FF/UMEC/VI significantly reduced the annual moderate/severe exacerbation rate compared with UMEC/VI in prior ICS users (29% reduction; P < 0.001), but only a numerical reduction was seen among prior ICS nonusers (12% reduction; P = 0.115). To minimize impact from ICS withdrawal, in an analysis excluding the first 30 days, FF/UMEC/VI continued to significantly reduce the annual on-treatment moderate/severe exacerbation rate (19%; P < 0.001) compared with UMEC/VI. The benefit of FF/UMEC/VI compared with UMEC/VI was seen for severe exacerbation rates, regardless of prior ICS use (prior ICS users, 35% reduction; P < 0.001; non-ICS users, 35% reduction; P = 0.018), and overall when excluding the first 30 days (29%; P < 0.001). Improvements from baseline with FF/UMEC/VI compared with UMEC/VI were also maintained throughout the study for both trough FEV1 and St. George's Respiratory Questionnaire, regardless of prior ICS use.Conclusions: These data support the important treatment effects of FF/UMEC/VI combination therapy on exacerbation reduction, lung function, and quality of life that do not appear to be related to abrupt ICS withdrawal.Clinical trial registered with www.clinicaltrials.gov (NCT02164513).

Reduction in All-Cause Mortality with Fluticasone Furoate/Umeclidinium/Vilanterol in Patients with Chronic Obstructive Pulmonary Disease.

Rationale: The IMPACT (Informing the Pathway of Chronic Obstructive Pulmonary Disease Treatment) trial demonstrated a significant reduction in all-cause mortality (ACM) risk with fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) versus UMEC/VI in patients with chronic obstructive pulmonary disease (COPD) at risk of future exacerbations. Five hundred seventy-four patients were censored in the original analysis owing to incomplete vital status information.Objectives: Report ACM and impact of stepping down therapy, following collection of additional vital status data.Methods: Patients were randomized 2:2:1 to FF/UMEC/VI 100/62.5/25 µg, FF/VI 100/25 µg, or UMEC/VI 62.5/25 µg following a run-in on their COPD therapies. Time to ACM was prespecified. Additional vital status data collection and subsequent analyses were performed post hoc.Measurements and Main Results: We report vital status data for 99.6% of the intention-to-treat population (n = 10,355), documenting 98 (2.36%) deaths on FF/UMEC/VI, 109 (2.64%) on FF/VI, and 66 (3.19%) on UMEC/VI. For FF/UMEC/VI, the hazard ratio for death was 0.72 (95% confidence interval, 0.53-0.99; P = 0.042) versus UMEC/VI and 0.89 (95% confidence interval, 0.67-1.16; P = 0.387) versus FF/VI. Independent adjudication confirmed lower rates of cardiovascular and respiratory death and death associated with the patient's COPD.Conclusions: In this secondary analysis of an efficacy outcome from the IMPACT trial, once-daily single-inhaler FF/UMEC/VI triple therapy reduced the risk of ACM versus UMEC/VI in patients with symptomatic COPD and a history of exacerbations.

The Importance of N186 in the Alpha-1-Antitrypsin Shutter Region Is Revealed by the Novel Bologna Deficiency Variant.

Alpha-1-antitrypsin (AAT) deficiency causes pulmonary disease due to decreased levels of circulating AAT and consequently unbalanced protease activity in the lungs. Deposition of specific AAT variants, such as the common Z AAT, within hepatocytes may also result in liver disease. These deposits are comprised of ordered polymers of AAT formed by an inter-molecular domain swap. The discovery and characterization of rare variants of AAT and other serpins have historically played a crucial role in the dissection of the structural mechanisms leading to AAT polymer formation. Here, we report a severely deficient shutter region variant, Bologna AAT (N186Y), which was identified in five unrelated subjects with different geographical origins. We characterized the new variant by expression in cellular models in comparison with known polymerogenic AAT variants. Bologna AAT showed secretion deficiency and intracellular accumulation as detergent-insoluble polymers. Extracellular polymers were detected in both the culture media of cells expressing Bologna AAT and in the plasma of a patient homozygous for this variant. Structural modelling revealed that the mutation disrupts the hydrogen bonding network in the AAT shutter region. These data support a crucial coordinating role for asparagine 186 and the importance of this network in promoting formation of the native structure.

Heteropolymerization of α-1-antitrypsin mutants in cell models mimicking heterozygosity.

The most common genotype associated with severe α-1-antitrypsin deficiency (AATD) is the Z homozygote. The Z variant (Glu342Lys) of α-1-antitrypsin (AAT) undergoes a conformational change and is retained within the endoplasmic reticulum (ER) of hepatocytes leading to the formation of ordered polymeric chains and inclusion bodies. Accumulation of mutated protein predisposes to cirrhosis whilst plasma AAT deficiency leads to emphysema. Increased risk of liver and lung disease has also been reported in heterozygous subjects who carry Z in association with the milder S allele (Glu264Val) or even with wild-type M. However, it is unknown whether Z AAT can co-polymerize with other AAT variants in vivo. We co-expressed two AAT variants, each modified by a different tag, in cell models that replicate AAT deficiency. We used pull-down assays to investigate interactions between co-expressed variants and showed that Z AAT forms heteropolymers with S and with the rare Mmalton (Phe52del) and Mwurzburg (Pro369Ser) mutants, and to a lesser extent with the wild-type protein. Heteropolymers were recognized by the 2C1 mAb that binds to Z polymers in vivo. There was increased intracellular accumulation of AAT variants when co-expressed with Z AAT, suggesting a dominant negative effect of the Z allele. The molecular interactions between S and Z AAT were confirmed by confocal microscopy showing their colocalization within dilated ER cisternae and by positivity in Proximity Ligation Assays. These results provide the first evidence of intracellular co-polymerization of AAT mutants and contribute to understanding the risk of liver disease in SZ and MZ heterozygotes.

Whole exome sequencing analysis in severe chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD), one of the leading causes of death worldwide, is substantially influenced by genetic factors. Alpha-1 antitrypsin deficiency demonstrates that rare coding variants of large effect can influence COPD susceptibility. To identify additional rare coding variants in patients with severe COPD, we conducted whole exome sequencing analysis in 2543 subjects from two family-based studies (Boston Early-Onset COPD Study and International COPD Genetics Network) and one case-control study (COPDGene). Applying a gene-based segregation test in the family-based data, we identified significant segregation of rare loss of function variants in TBC1D10A and RFPL1 (P-value < 2x10-6), but were unable to find similar variants in the case-control study. In single-variant, gene-based and pathway association analyses, we were unable to find significant findings that replicated or were significant in meta-analysis. However, we found that the top results in the two datasets were in proximity to each other in the protein-protein interaction network (P-value = 0.014), suggesting enrichment of these results for similar biological processes. A network of these association results and their neighbors was significantly enriched in the transforming growth factor beta-receptor binding and cilia-related pathways. Finally, in a more detailed examination of candidate genes, we identified individuals with putative high-risk variants, including patients harboring homozygous mutations in genes associated with cutis laxa and Niemann-Pick Disease Type C. Our results likely reflect heterogeneity of genetic risk for COPD along with limitations of statistical power and functional annotation, and highlight the potential of network analysis to gain insight into genetic association studies.

The effect of exacerbation history on outcomes in the IMPACT trial.

IMPACT, a 52-week, randomised, double-blind trial, assessed the efficacy and safety of fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) triple therapy versus FF/VI or UMEC/VI in patients with symptomatic COPD and a history of exacerbations.Subgroup analyses assessed whether the efficacy of FF/UMEC/VI versus FF/VI or UMEC/VI and UMEC/VI versus FF/VI varies according to prior exacerbation history, and the combined effects of exacerbation history and blood eosinophil counts. Three subgroups were defined: single moderate (1 moderate/no severe; n=3056 (30%)), frequent moderate (≥2 moderate/no severe; n=4628 (45%)) and severe (≥1 severe/any moderate; n=2671 (26%)). End-points included annual on-treatment moderate/severe exacerbation rate (pre-specified), lung function and health status (both post-hoc).Moderate/severe exacerbation rates (reduction % (95% CI)) were reduced in the FF/UMEC/VI group versus FF/VI (single moderate 20% (10-29), frequent moderate 11% (2-19), severe 17% (7-26)) and versus UMEC/VI (single moderate 18% (5-29), frequent moderate 29% (21-37), severe 26% (14-35)). Moderate/severe exacerbation rates were reduced in the FF/VI group versus UMEC/VI in the frequent moderate subgroup; a numerical reduction was observed in the severe subgroup (single moderate 2% (-12-18), frequent moderate 21% (11-29), severe 11% (-3-22)). Moderate/severe exacerbation rates were lower in the FF/VI group compared with UMEC/VI in patients with higher eosinophil counts. FF/UMEC/VI improved lung function and health status versus both dual therapies irrespective of exacerbation subgroup. UMEC/VI improved lung function versus FF/VI in all subgroups.Triple therapy was more effective than dual regardless of exacerbation history, consistent with results in the intent-to-treat population. Comparisons between dual therapies were influenced by prior exacerbation history and eosinophil counts.

Single-inhaler triple therapy fluticasone furoate/umeclidinium/vilanterol versus fluticasone furoate/vilanterol and umeclidinium/vilanterol in patients with COPD: results on cardiovascular safety from the IMPACT trial.

BACKGROUND: This analysis of the IMPACT study assessed the cardiovascular (CV) safety of single-inhaler triple therapy with fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) versus FF/VI and UMEC/VI dual therapy. METHODS: IMPACT was a 52-week, randomized, double-blind, multicenter Phase III study comparing the efficacy and safety of FF/UMEC/VI 100/62.5/25 mcg with FF/VI 100/25 mcg or UMEC/VI 62.5/25 mcg in patients ≥40 years of age with symptomatic chronic obstructive pulmonary disease (COPD) and ≥1 moderate/severe exacerbation in the previous year. The inclusion criteria for the study were intentionally designed to permit the enrollment of patients with significant concurrent CV disease/risk. CV safety assessments included proportion of patients with and exposure-adjusted rates of on-treatment CV adverse events of special interest (CVAESI) and major adverse cardiac events (MACE), as well as time-to-first (TTF) CVAESI, and TTF CVAESI resulting in hospitalization/prolonged hospitalization or death. RESULTS: Baseline CV risk factors were similar across treatment groups. Overall, 68% of patients (n = 7012) had ≥1 CV risk factor and 40% (n = 4127) had ≥2. At baseline, 29% of patients reported a current/past cardiac disorder and 58% reported a current/past vascular disorder. The proportion of patients with on-treatment CVAESI was 11% for both FF/UMEC/VI and UMEC/VI, and 10% for FF/VI. There was no statistical difference for FF/UMEC/VI versus FF/VI or UMEC/VI in TTF CVAESI (hazard ratio [HR]: 0.98, 95% confidence interval [CI]: 0.85, 1.11; p = 0.711 and HR: 0.92, 95% CI: 0.78, 1.08; p = 0.317, respectively) nor TTF CVAESI leading to hospitalization/prolonged hospitalization or death (HR: 1.19, 95% CI: 0.93, 1.51; p = 0.167 and HR: 0.96, 95% CI: 0.72, 1.27; p = 0.760, respectively). On-treatment MACE occurred in ≤3% of patients across treatment groups, with similar prevalence and rates between treatments. CONCLUSIONS: In a symptomatic COPD population with a history of exacerbations and a high rate of CV disease/risk, the proportion of patients with CVAESI and MACE was 10-11% and 1-3%, respectively, across treatment arms, and the risk of CVAESI was low and similar across treatment arms. There was no statistically significant increased CV risk associated with the use of FF/UMEC/VI versus FF/VI or UMEC/VI, and UMEC/VI versus FF/VI. TRIAL REGISTRATION: NCT02164513 (GSK study number CTT116855).

Heme metabolism genes Downregulated in COPD Cachexia.

INTRODUCTION: Cachexia contributes to increased mortality and reduced quality of life in Chronic Obstructive Pulmonary Disease (COPD) and may be associated with underlying gene expression changes. Our goal was to identify differential gene expression signatures associated with COPD cachexia in current and former smokers. METHODS: We analyzed whole-blood gene expression data from participants with COPD in a discovery cohort (COPDGene, N = 400) and assessed replication (ECLIPSE, N = 114). To approximate the consensus definition using available criteria, cachexia was defined as weight-loss > 5% in the past 12 months or low body mass index (BMI) (< 20 kg/m2) and 1/3 criteria: decreased muscle strength (six-minute walk distance < 350 m), anemia (hemoglobin < 12 g/dl), and low fat-free mass index (FFMI) (< 15 kg/m2 among women and < 17 kg/m2 among men) in COPDGene. In ECLIPSE, cachexia was defined as weight-loss > 5% in the past 12 months or low BMI and 3/5 criteria: decreased muscle strength, anorexia, abnormal biochemistry (anemia or high c-reactive protein (> 5 mg/l)), fatigue, and low FFMI. Differential gene expression was assessed between cachectic and non-cachectic subjects, adjusting for age, sex, white blood cell counts, and technical covariates. Gene set enrichment analysis was performed using MSigDB. RESULTS: The prevalence of COPD cachexia was 13.7% in COPDGene and 7.9% in ECLIPSE. Fourteen genes were differentially downregulated in cachectic versus non-cachectic COPD patients in COPDGene (FDR < 0.05) and ECLIPSE (FDR < 0.05). DISCUSSION: Several replicated genes regulating heme metabolism were downregulated among participants with COPD cachexia. Impaired heme biosynthesis may contribute to cachexia development through free-iron buildup and oxidative tissue damage.

It's more than low BMI: prevalence of cachexia and associated mortality in COPD.

BACKGROUND: Cachexia is associated with increased mortality risk among chronic obstructive pulmonary disease (COPD) patients. However, low body mass index (BMI) as opposed to cachexia is often used, particularly when calculating the BODE (BMI, Obstruction, Dyspnea and Exercise) index. For this reason, we examined mortality using a consensus definition and a weight-loss definition of cachexia among COPD cases and compared two new COPD severity indices with BODE. METHODS: In the current report, the consensus definition for cachexia incorporated weight-loss > 5% in 12-months or low BMI in addition to 3/5 of decreased muscle strength, fatigue, anorexia, low FFMI and inflammation. The weight-loss definition incorporated weight-loss > 5% or weight-loss > 2% (if low BMI) in 12-months. The low BMI component in BODE was replaced with the consensus definition to create the CODE (Consensus cachexia, Obstruction, Dyspnea and Exercise) index and the weight-loss definition to create the WODE (Weight loss, Obstruction, Dyspnea and Exercise) index. Mortality was assessed using Kaplan-Meier survival and Cox Regression. Performance of models was compared using C-statistics. RESULTS: Among 1483 COPD cases, the prevalences of cachexia by the consensus and weight-loss definitions were 4.7 and 10.4%, respectively. Cachectic patients had a greater than three-fold increased mortality by either the consensus or the weight-loss definition of cachexia independent of BMI and lung function. The CODE index predicted mortality slightly more accurately than the BODE and WODE indices. CONCLUSIONS: Cachexia is associated with increased mortality among COPD patients. Monitoring cachexia using weight-loss criteria is relatively simple and predictive of mortality among COPD cases who may be missed if only low BMI is used.

Common and Rare Variants Genetic Association Analysis of Cigarettes per Day Among Ever-Smokers in Chronic Obstructive Pulmonary Disease Cases and Controls.

INTRODUCTION: Cigarette smoking is a major environmental risk factor for many diseases, including chronic obstructive pulmonary disease (COPD). There are shared genetic influences on cigarette smoking and COPD. Genetic risk factors for cigarette smoking in cohorts enriched for COPD are largely unknown. METHODS: We performed genome-wide association analyses for average cigarettes per day (CPD) across the Genetic Epidemiology of COPD (COPDGene) non-Hispanic white (NHW) (n = 6659) and African American (AA) (n = 3260), GenKOLS (the Genetics of Chronic Obstructive Lung Disease) (n = 1671), and ECLIPSE (the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) (n = 1942) cohorts. In addition, we performed exome array association analyses across the COPDGene NHW and AA cohorts. We considered analyses across the entire cohort and stratified by COPD case-control status. RESULTS: We identified genome-wide significant associations for CPD on chromosome 15q25 across all cohorts (lowest p = 1.78 × 10-15), except in the COPDGene AA cohort alone. Previously reported associations on chromosome 19 had suggestive and directionally consistent associations (RAB4, p = 1.95 × 10-6; CYP2A7, p = 7.50 × 10-5; CYP2B6, p = 4.04 × 10-4). When we stratified by COPD case-control status, single nucleotide polymorphisms on chromosome 15q25 were nominally associated with both NHW COPD cases (ß = 0.11, p = 5.58 × 10-4) and controls (ß = 0.12, p = 3.86 × 10-5) For the gene-based exome array association analysis of rare variants, there were no exome-wide significant associations. For these previously replicated associations, the most significant results were among COPDGene NHW subjects for CYP2A7 (p = 5.2 × 10-4). CONCLUSIONS: In a large genome-wide association study of both common variants and a gene-based association of rare coding variants in ever-smokers, we found genome-wide significant associations on chromosome 15q25 with CPD for common variants, but not for rare coding variants. These results were directionally consistent among COPD cases and controls. IMPLICATIONS: We examined both common and rare coding variants associated with CPD in a large population of heavy smokers with and without COPD of NHW and AA descent. We replicated genome-wide significant associations on chromosome 15q25 with CPD for common variants among NHW subjects, but not for rare variants. We demonstrated for the first time that common variants on chromosome 15q25 associated with CPD are similar among COPD cases and controls. Previously reported associations on chromosome 19 showed suggestive and directionally consistent associations among common variants (RAB4, CYP2A7, and CYP2B6) and for rare variants (CYP2A7) among COPDGene NHW subjects. Although the genetic effect sizes for these single nucleotide polymorphisms on chromosome 15q25 are modest, we show that this creates a substantial smoking burden over the lifetime of a smoker.

hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in α1-antitrypsin deficiency.

BACKGROUND & AIMS: α1-Antitrypsin deficiency (A1ATD) is an autosomal recessive disorder caused by mutations in the SERPINA1 gene. Individuals with the Z variant (Gly342Lys) retain polymerised protein in the endoplasmic reticulum (ER) of their hepatocytes, predisposing them to liver disease. The concomitant lack of circulating A1AT also causes lung emphysema. Greater insight into the mechanisms that link protein misfolding to liver injury will facilitate the design of novel therapies. METHODS: Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes provide a novel approach to interrogate the molecular mechanisms of A1ATD because of their patient-specific genetic architecture and reflection of human physiology. To that end, we utilised patient-specific hiPSC hepatocyte-like cells (ZZ-HLCs) derived from an A1ATD (ZZ) patient, which faithfully recapitulated key aspects of the disease at the molecular and cellular level. Subsequent functional and "omics" comparisons of these cells with their genetically corrected isogenic-line (RR-HLCs) and primary hepatocytes/human tissue enabled identification of new molecular markers and disease signatures. RESULTS: Our studies showed that abnormal A1AT polymer processing (immobilised ER components, reduced luminal protein mobility and disrupted ER cisternae) occurred heterogeneously within hepatocyte populations and was associated with disrupted mitochondrial structure, presence of the oncogenic protein AKR1B10 and two upregulated molecular clusters centred on members of inflammatory (IL-18 and Caspase-4) and unfolded protein response (Calnexin and Calreticulin) pathways. These results were validated in a second patient-specific hiPSC line. CONCLUSIONS: Our data identified novel pathways that potentially link the expression of Z A1AT polymers to liver disease. These findings could help pave the way towards identification of new therapeutic targets for the treatment of A1ATD. LAY SUMMARY: This study compared the gene expression and protein profiles of healthy liver cells and those affected by the inherited disease α1-antitrypsin deficiency. This approach identified specific factors primarily present in diseased samples which could provide new targets for drug development. This study also demonstrates the interest of using hepatic cells generated from human-induced pluripotent stem cells to model liver disease in vitro for uncovering new mechanisms with clinical relevance.