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Angiopoietin-like 4 (ANGPTL4) is an endogenous inhibitor of lipoprotein lipase that modulates lipid levels, coronary atherosclerosis risk, and nutrient partitioning. We hypothesize that loss of ANGPTL4 function might improve glucose homeostasis and decrease risk of type 2 diabetes (T2D). We investigate protein-altering variants in ANGPTL4 among 58,124 participants in the DiscovEHR human genetics study, with follow-up studies in 82,766 T2D cases and 498,761 controls. Carriers of p.E40K, a variant that abolishes ANGPTL4 ability to inhibit lipoprotein lipase, have lower odds of T2D (odds ratio 0.89, 95% confidence interval 0.85-0.92, p = 6.3 × 10 -10 ), lower fasting glucose, and greater insulin sensitivity. Predicted loss-of-function variants are associated with lower odds of T2D among 32,015 cases and 84,006 controls (odds ratio 0.71, 95% confidence interval 0.49-0.99, p = 0.041). Functional studies in Angptl4-deficient mice confirm improved insulin sensitivity and glucose homeostasis. In conclusion, genetic inactivation of ANGPTL4 is associated with improved glucose homeostasis and reduced risk of T2D.

Background: Loss-of-function variants in the angiopoietin-like 3 gene (ANGPTL3) have been associated with decreased plasma levels of triglycerides, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol. It is not known whether such variants or therapeutic antagonism of ANGPTL3 are associated with a reduced risk of atherosclerotic cardiovascular disease., Methods: We sequenced the exons of ANGPTL3 in 58,335 participants in the DiscovEHR human genetics study. We performed tests of association for loss-of-function variants in ANGPTL3 with lipid levels and with coronary artery disease in 13,102 case patients and 40,430 controls from the DiscovEHR study, with follow-up studies involving 23,317 case patients and 107,166 controls from four population studies. We also tested the effects of a human monoclonal antibody, evinacumab, against Angptl3 in dyslipidemic mice and against ANGPTL3 in healthy human volunteers with elevated levels of triglycerides or LDL cholesterol., Results: In the DiscovEHR study, participants with heterozygous loss-of-function variants in ANGPTL3 had significantly lower serum levels of triglycerides, HDL cholesterol, and LDL cholesterol than participants without these variants. Loss-of-function variants were found in 0.33% of case patients with coronary artery disease and in 0.45% of controls (adjusted odds ratio, 0.59; 95% confidence interval, 0.41 to 0.85; P=0.004). These results were confirmed in the follow-up studies. In dyslipidemic mice, inhibition of Angptl3 with evinacumab resulted in a greater decrease in atherosclerotic lesion area and necrotic content than a control antibody. In humans, evinacumab caused a dose-dependent placebo-adjusted reduction in fasting triglyceride levels of up to 76% and LDL cholesterol levels of up to 23%., Conclusions: Genetic and therapeutic antagonism of ANGPTL3 in humans and of Angptl3 in mice was associated with decreased levels of all three major lipid fractions and decreased odds of atherosclerotic cardiovascular disease. (Funded by Regeneron Pharmaceuticals and others; ClinicalTrials.gov number, NCT01749878 .).

The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated β-galactosidase (SA-β-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema.

The DiscovEHR collaboration between the Regeneron Genetics Center and Geisinger Health System couples high-throughput sequencing to an integrated health care system using longitudinal electronic health records (EHRs). We sequenced the exomes of 50,726 adult participants in the DiscovEHR study to identify ~4.2 million rare single-nucleotide variants and insertion/deletion events, of which ~176,000 are predicted to result in a loss of gene function. Linking these data to EHR-derived clinical phenotypes, we find clinical associations supporting therapeutic targets, including genes encoding drug targets for lipid lowering, and identify previously unidentified rare alleles associated with lipid levels and other blood level traits. About 3.5% of individuals harbor deleterious variants in 76 clinically actionable genes. The DiscovEHR data set provides a blueprint for large-scale precision medicine initiatives and genomics-guided therapeutic discovery., (Copyright © 2016, American Association for the Advancement of Science.)

Background: Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible airflow limitation in response to inhalation of noxious stimuli, such as cigarette smoke. However, only 15-20 % smokers manifest COPD, suggesting a role for genetic predisposition. Although genome-wide association studies have identified common genetic variants that are associated with susceptibility to COPD, effect sizes of the identified variants are modest, as is the total heritability accounted for by these variants. In this study, an extreme phenotype exome sequencing study was combined with in vitro modeling to identify COPD candidate genes., Results: We performed whole exome sequencing of 62 highly susceptible smokers and 30 exceptionally resistant smokers to identify rare variants that may contribute to disease risk or resistance to COPD. This was a cross-sectional case-control study without therapeutic intervention or longitudinal follow-up information. We identified candidate genes based on rare variant analyses and evaluated exonic variants to pinpoint individual genes whose function was computationally established to be significantly different between susceptible and resistant smokers. Top scoring candidate genes from these analyses were further filtered by requiring that each gene be expressed in human bronchial epithelial cells (HBECs). A total of 81 candidate genes were thus selected for in vitro functional testing in cigarette smoke extract (CSE)-exposed HBECs. Using small interfering RNA (siRNA)-mediated gene silencing experiments, we showed that silencing of several candidate genes augmented CSE-induced cytotoxicity in vitro., Conclusions: Our integrative analysis through both genetic and functional approaches identified two candidate genes (TACC2 and MYO1E) that augment cigarette smoke (CS)-induced cytotoxicity and, potentially, COPD susceptibility.

Our previous studies showed that cigarette smokers who are exposed to wood smoke (WS) are at an increased risk for chronic bronchitis and reduced lung function. The present study was undertaken to determine the mechanisms for WS-induced adverse effects. We studied the effect of WS exposure using four cohorts of mice. C57Bl/6 mice were exposed for 4 or 12 weeks to filtered air, to 10 mg/m(3) WS for 2 h/d, to 250 mg/m(3) cigarette smoke (CS) for 6 h/d, or to CS followed by WS (CW). Inflammation was absent in the filtered air and WS groups, but enhanced by twofold in the bronchoalveolar lavage of the CW compared with CS group as measured by neutrophil numbers and levels of the neutrophil chemoattractant, keratinocyte-derived chemokine. The levels of the anti-inflammatory lipoxin, lipoxin A4, were reduced by threefold along with cyclo-oxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1 in airway epithelial cells and PGE2 levels in the bronchoalveolar lavage of CW compared with CS mice. We replicated, in primary human airway epithelial cells, the changes observed in mice. Immunoprecipitations showed that WS blocked the interaction of aryl hydrocarbon receptor (AHR) with AHR nuclear transporter to reduce expression of COX-2 and mPGES-1 by increasing expression of AHR repressor (AHRR). Collectively, these studies show that exposure to low concentrations of WS enhanced CS-induced inflammation by inducing AHRR expression to suppress AHR, COX-2, and mPGES-1 expression, and levels of PGE2 and lipoxin A4. Therefore, AHRR is a potential therapeutic target for WS-associated exacerbations of CS-induced inflammation.

Background: Acrolein is a ubiquitous environmental hazard to human health. Acrolein has been reported to activate the DNA damage response and induce apoptosis. However, little is known about the effects of acrolein on cellular senescence., Objectives: We examined whether acrolein induces cellular senescence in cultured normal human lung fibroblasts (NHLF)., Methods: We cultured NHLF in the presence or absence of acrolein and determined the effects of acrolein on cell proliferative capacity, senescence-associated β-galactosidase activity, the known senescence-inducing pathways (e.g., p53, p21), and telomere length., Results: We found that acrolein induced cellular senescence by increasing both p53 and p21. The knockdown of p53 mediated by small interfering RNA (siRNA) attenuated acrolein-induced cellular senescence. Acrolein decreased Werner's syndrome protein (WRN), a member of the RecQ helicase family involved in DNA repair and telomere maintenance. Acrolein-induced down-regulation of WRN protein was rescued by p53 knockdown or proteasome inhibition. Finally, we found that acrolein accelerated p53-mediated telomere shortening., Conclusions: These results suggest that acrolein induces p53-mediated cellular senescence accompanied by enhanced telomere attrition and WRN protein down-regulation.

Rationale: Sepsis is a leading cause of morbidity and mortality. Currently, early diagnosis and the progression of the disease are difficult to make. The integration of metabolomic and transcriptomic data in a primate model of sepsis may provide a novel molecular signature of clinical sepsis., Objectives: To develop a biomarker panel to characterize sepsis in primates and ascertain its relevance to early diagnosis and progression of human sepsis., Methods: Intravenous inoculation of Macaca fascicularis with Escherichia coli produced mild to severe sepsis, lung injury, and death. Plasma samples were obtained before and after 1, 3, and 5 days of E. coli challenge and at the time of killing. At necropsy, blood, lung, kidney, and spleen samples were collected. An integrative analysis of the metabolomic and transcriptomic datasets was performed to identify a panel of sepsis biomarkers., Measurements and Main Results: The extent of E. coli invasion, respiratory distress, lethargy, and mortality was dependent on the bacterial dose. Metabolomic and transcriptomic changes characterized severe infections and death, and indicated impaired mitochondrial, peroxisomal, and liver functions. Analysis of the pulmonary transcriptome and plasma metabolome suggested impaired fatty acid catabolism regulated by peroxisome-proliferator activated receptor signaling. A representative four-metabolite model effectively diagnosed sepsis in primates (area under the curve, 0.966) and in two human sepsis cohorts (area under the curve, 0.78 and 0.82)., Conclusions: A model of sepsis based on reciprocal metabolomic and transcriptomic data was developed in primates and validated in two human patient cohorts. It is anticipated that the identified parameters will facilitate early diagnosis and management of sepsis.

Background: Whether Native American ancestry (NAA) is associated with COPD or lung function in a racially admixed Hispanic population is unknown., Methods: We recruited 578 Costa Ricans with and without COPD into a hybrid case-control/family-based cohort, including 316 members of families of index case subjects. All participants completed questionnaires and spirometry and gave a blood sample for DNA extraction. Genome-wide genotyping was conducted with the Illumina Human610-Quad and HumanOmniExpress BeadChip kits (Illumina Inc), and individual ancestral proportions were estimated from these genotypic data and reference panels. For unrelated individuals, linear or logistic regression was used for the analysis of NAA and COPD (GOLD [Global Initiative for Chronic Obstructive Lung Disease] stage II or greater) or lung function. For extended families, linear mixed models and generalized estimating equations were used for the analysis. All models were adjusted for age, sex, educational level, and smoking behavior; models for FEV1 were also adjusted for height., Results: The average proportion of European, Native American, and African ancestry among participants was 62%, 35%, and 3%, respectively. After adjustment for current smoking and other covariates, NAA was inversely associated with COPD (OR per 10% increment, 0.55; 95% CI, 0.41-0.75) but positively associated with FEV1, FVC, and FEV1/FVC. After additional adjustment for pack-years of smoking, the association between NAA and COPD or lung function measures was slightly attenuated. We found that about 31% of the estimated effect of NAA on COPD is mediated by pack-years of smoking., Conclusions: NAA is inversely associated with COPD but positively associated with FEV1 or FVC in Costa Ricans. Ancestral effects on smoking behavior partly explain the findings for COPD but not for FEV1 or FVC.

Aldehyde dehydrogenase 3A1 (ALDH3A1), an ALDH superfamily member, catalyzes the oxidation of reactive aldehydes, highly toxic components of cigarette smoke (CS). Even so, the role of ALDH3A1 in CS-induced cytotoxicity and DNA damage has not been examined. Among all of the ALDH superfamily members, ALDH3A1 mRNA levels showed the greatest induction in response to CS extract (CSE) exposure of primary human bronchial epithelial cells (HBECs). ALDH3A1 protein accumulation was accompanied by increased ALDH enzymatic activity in CSE-exposed immortalized HBECs. The effects of overexpression or suppression of ALDH3A1 on CSE-induced cytotoxicity and DNA damage (γH2AX) were evaluated in cultured immortalized HBECs. Enforced expression of ALDH3A1 attenuated cytotoxicity and downregulated γH2AX. SiRNA-mediated suppression of ALDH3A1 blocked ALDH enzymatic activity and augmented cytotoxicity in CSE-exposed cells. Our results suggest that the availability of ALDH3A1 is important for cell survival against CSE in HBECs., (Published by Elsevier Inc.)

Background: Chronic mucous hypersecretion (CMH) contributes to COPD exacerbations and increased risk for lung cancer. Because methylation of gene promoters in sputum has been shown to be associated with lung cancer risk, we tested whether such methylation was more common in persons with CMH., Methods: Eleven genes commonly silenced by promoter methylation in lung cancer and associated with cancer risk were selected. Methylation specific PCR (MSP) was used to profile the sputum of 900 individuals in the Lovelace Smokers Cohort (LSC). Replication was performed in 490 individuals from the Pittsburgh Lung Screening Study (PLuSS)., Results: CMH was significantly associated with an overall increased number of methylated genes, with SULF2 methylation demonstrating the most consistent association. The association between SULF2 methylation and CMH was significantly increased in males but not in females both in the LSC and PLuSS (OR = 2.72, 95% CI = 1.51-4.91, p = 0.001 and OR = 2.97, 95% CI = 1.48-5.95, p = 0.002, respectively). Further, the association between methylation and CMH was more pronounced among 139 male former smokers with persistent CMH compared to current smokers (SULF2; OR = 3.65, 95% CI = 1.59-8.37, p = 0.002)., Conclusions: These findings demonstrate that especially male former smokers with persistent CMH have markedly increased promoter methylation of lung cancer risk genes and potentially could be at increased risk for lung cancer.

The BODE index was developed as a prognostic mortality risk tool for persons with chronic obstructive pulmonary disease (COPD). It incorporates 4 measures: body mass index, lung obstruction, dyspnea, and exercise capacity. The intent of this study was to examine how well a BODE-like index constructed using a simpler lung function measure, peak expiratory flow, in combination with physical functioning and symptom information more readily found in survey data (a quasi-BODE index), performs in identifying persons at higher risk of mortality and whether it may be extended as an assessment of mortality risk to persons without diagnosed COPD. Using US national survey data from the Health Retirement Study for 2006-2010, each unit increase in the quasi-BODE index score was associated with a multiplicative 50% increase in mortality risk (odds ratio = 1.50, 95% confidence interval: 1.41, 1.59). The quasi-BODE index is a multidimensional health status instrument based on the BODE index, which is a good predictor of mortality. The quasi-BODE index was compiled using simple measures of physical and respiratory function. It is a potentially useful prognostic instrument for older adult populations with or without COPD, including those with severe physical limitations, particularly when combined with demographic factors and comorbid conditions.

The estimation of genetic ancestry in human populations has important applications in medical genetic studies. Genetic ancestry is used to control for population stratification in genetic association studies, and is used to understand the genetic basis for ethnic differences in disease susceptibility. In this review, we present an overview of genetic ancestry estimation in human disease studies, followed by a review of popular softwares and methods used for this estimation.

Rationale: Traditional genome-wide association studies (GWASs) of large cohorts of subjects with chronic obstructive pulmonary disease (COPD) have successfully identified novel candidate genes, but several other plausible loci do not meet strict criteria for genome-wide significance after correction for multiple testing., Objectives: The authors hypothesise that by applying unbiased weights derived from unique populations we can identify additional COPD susceptibility loci. Methods The authors performed a homozygosity haplotype analysis on a group of subjects with and without COPD to identify regions of conserved homozygosity haplotype (RCHHs). Weights were constructed based on the frequency of these RCHHs in case versus controls, and used to adjust the p values from a large collaborative GWAS of COPD., Results: The authors identified 2318 RCHHs, of which 576 were significantly (p<0.05) over-represented in cases. After applying the weights constructed from these regions to a collaborative GWAS of COPD, the authors identified two single nucleotide polymorphisms (SNPs) in a novel gene (fibroblast growth factor-7 (FGF7)) that gained genome-wide significance by the false discovery rate method. In a follow-up analysis, both SNPs (rs12591300 and rs4480740) were significantly associated with COPD in an independent population (combined p values of 7.9E-7 and 2.8E-6, respectively). In another independent population, increased lung tissue FGF7 expression was associated with worse measures of lung function., Conclusion: Weights constructed from a homozygosity haplotype analysis of an isolated population successfully identify novel genetic associations from a GWAS on a separate population. This method can be used to identify promising candidate genes that fail to meet strict correction for multiple testing.

Rationale: The epidemiology of cigarette smoking-related chronic obstructive pulmonary disease (COPD) is not well characterized in Hispanics in the United States. Understanding how ethnicity influences COPD is important for a number of reasons, from informing public health policies to dissecting the genetic and environmental effects that contribute to disease., Objectives: The present study assessed differences in risk between Hispanics and non-Hispanic whites for longitudinal and cross-sectional COPD phenotypes. Genetic ancestry was used to verify findings based on self-reported ethnicity. Hispanics in New Mexico are primarily differentiated from non-Hispanic whites by their proportion of Native American ancestry., Methods: The study was performed in a New Mexican cohort of current and former smokers. Self-reported Hispanic and non-Hispanic white ethnicity was validated by defining genetic ancestry proportions at the individual level using 48 single-nucleotide polymorphism markers. Self-reported ethnicity and genetic ancestry were independently used to assess associations with cross-sectional and longitudinal measures of lung function. Multivariable models were adjusted for indicators of smoking behavior., Measurements and Main Results: Self-reported Hispanic ethnicity was significantly associated with lower odds of COPD (odds ratio, 0.49; 95% confidence interval, 0.35-0.71; P = 0.007), and this protection was validated by the observation that Hispanic smokers have reduced risk of rapid decline in lung function (odds ratio, 0.48; 95% confidence interval, 0.30-0.78; P = 0.003). Similar findings were noted when Native American genetic ancestry proportions were used as predictors instead of self-report of Hispanic ethnicity., Conclusions: Hispanic ethnicity is inversely associated with cross-sectional and longitudinal spirometric COPD phenotypes even after adjustment for smoking. Native American genetic ancestry may account for this "Hispanic protection."

We describe a bead-based, multiplexed, oligonucleotide ligation assay (OLA) performed on the Luminex flow cytometer. Differences between this method and those previously reported include the use of far fewer beads and the use of a universal oligonucleotide for signal detection. These innovations serve to significantly reduce the cost of the assay, while maintaining robustness and accuracy. Comparisons are made between the Luminex OLA and both pyrosequencing and direct sequencing. Experiments to assess conversion rates, call rates, and concordance across technical replicates are also presented.

The discovery of double-stranded RNA interference (dsRNAi) in Trypanosoma brucei provides a convenient method to generate knockout phenotypes in this protozoan parasite [Ngo H, Tschudi C, Gull K, Ullu E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA 1998;95:14687-14692]. The presence of double-stranded RNA (dsRNA) dominantly silences gene expression in a sequence-specific manner by causing the corresponding endogenous RNA to be degraded. To simplify the generation of knockout phenotypes in T. brucei via dsRNAi, we used two promoters arranged as an inverted repeat on a plasmid. This promoter arrangement generates transcripts of both strands of DNA inserted between the promoters, which then form dsRNA. We have used plasmids encoding either two T. brucei ribosomal RNA promoters or two bacteriophage T7 promoters to interfere with expression of alpha-tubulin (TUB), green fluorescent protein (GFP), paraflagellar rod protein A (PFRA), flagellum-adhesion glycoprotein 1 (FLA1), and histone 2B (H2B) in T. brucei. We show here that FLA1 is required for flagellar attachment in T. brucei and that H2B is required for parasite growth. Thus, the two-promoter approach efficiently generates dsRNAi in T. brucei and can be used to produce both specific and random knockout phenotypes in T. brucei. This approach should be useful in generating knockout phenotypes in other kinetoplastid parasites including Trypanosoma cruzi and Leishmania.