Age-associated differences in the human lung extracellular matrix.

Extracellular matrix (ECM) remodeling has been associated with chronic lung diseases. However, information about specific age-associated differences in lung ECM is currently limited. In this study, we aimed to identify and localize age-associated ECM differences in human lungs using comprehensive transcriptomic, proteomic, and immunohistochemical analyses. Our previously identified age-associated gene expression signature of the lung was re-analyzed limiting it to an aging signature based on 270 control patients (37-80 years) and focused on the Matrisome core geneset using geneset enrichment analysis. To validate the age-associated transcriptomic differences on protein level, we compared the age-associated ECM genes (false discovery rate, FDR < 0.05) with a profile of age-associated proteins identified from a lung tissue proteomics dataset from nine control patients (49-76 years) (FDR < 0.05). Extensive immunohistochemical analysis was used to localize and semi-quantify the age-associated ECM differences in lung tissues from 62 control patients (18-82 years). Comparative analysis of transcriptomic and proteomic data identified seven ECM proteins with higher expression with age at both gene and protein levels: COL1A1, COL6A1, COL6A2, COL14A1, FBLN2, LTBP4, and LUM. With immunohistochemistry, we demonstrated higher protein levels with age for COL6A2 in whole tissue, parenchyma, airway wall, and blood vessel, for COL14A1 and LUM in bronchial epithelium, and COL1A1 in lung parenchyma. Our study revealed that higher age is associated with lung ECM remodeling, with specific differences occurring in defined regions within the lung. These differences may affect lung structure and physiology with aging and as such may increase susceptibility to developing chronic lung diseases.NEW & NOTEWORTHY We identified seven age-associated extracellular matrix (ECM) proteins, i.e., COL1A1, COL6A1, COL6A2 COL14A1, FBLN2, LTBP4, and LUM with higher transcript and protein levels in human lung tissue with age. Extensive immunohistochemical analysis revealed significant age-associated differences for COL6A2 in whole tissue, parenchyma, airway wall, and vessel, for COL14A1 and LUM in bronchial epithelium, and COL1A1 in parenchyma. Our findings lay a new foundation for the investigation of ECM differences in age-associated chronic lung diseases.

Aryl hydrocarbon receptor deficiency causes the development of chronic obstructive pulmonary disease through the integration of multiple pathogenic mechanisms.

Emphysema, a component of chronic obstructive pulmonary disease (COPD), is characterized by irreversible alveolar destruction that results in a progressive decline in lung function. This alveolar destruction is caused by cigarette smoke, the most important risk factor for COPD. Only 15%-20% of smokers develop COPD, suggesting that unknown factors contribute to disease pathogenesis. We postulate that the aryl hydrocarbon receptor (AHR), a receptor/transcription factor highly expressed in the lungs, may be a new susceptibility factor whose expression protects against COPD. Here, we report that Ahr-deficient mice chronically exposed to cigarette smoke develop airspace enlargement concomitant with a decline in lung function. Chronic cigarette smoke exposure also increased cleaved caspase-3, lowered SOD2 expression, and altered MMP9 and TIMP-1 levels in Ahr-deficient mice. We also show that people with COPD have reduced expression of pulmonary and systemic AHR, with systemic AHR mRNA levels positively correlating with lung function. Systemic AHR was also lower in never-smokers with COPD. Thus, AHR expression protects against the development of COPD by controlling interrelated mechanisms involved in the pathogenesis of this disease. This study identifies the AHR as a new, central player in the homeostatic maintenance of lung health, providing a foundation for the AHR as a novel therapeutic target and/or predictive biomarker in chronic lung disease.

Rhinovirus and asthma: Challenges and opportunities.

Human rhinoviruses (RVs) are the primary aetiological agent of the common cold. Generally, the associated infection is mild and self-limiting, but may also be associated with bronchiolitis in infants, pneumonia in the immunocompromised and exacerbation in patients with pulmonary conditions such as asthma or chronic obstructive pulmonary disease. Viral infection accounts for as many as two thirds of asthma exacerbations in children and more than half in adults. Allergy and asthma are major risk factors for more frequent and severe RV-related illnesses. The prevalence of RV-induced wheezing will likely continue to increase given that asthma affects a significant proportion of the population, with allergic asthma accounting for the majority. Several new respiratory viruses and their subgroups have been discovered, with various degrees of relevance. This review will focus on RV infection in the context of the epidemiologic evidence, genetic variability, pathobiology, clinical studies in the context of asthma, differences with other viruses including COVID-19 and current treatment interventions.

Phenotypic and functional translation of IL33 genetics in asthma.

BACKGROUND: Asthma is a complex disease with multiple phenotypes that may differ in disease pathobiology and treatment response. IL33 single nucleotide polymorphisms (SNPs) have been reproducibly associated with asthma. IL33 levels are elevated in sputum and bronchial biopsies of patients with asthma. The functional consequences of IL33 asthma SNPs remain unknown. OBJECTIVE: This study sought to determine whether IL33 SNPs associate with asthma-related phenotypes and with IL33 expression in lung or bronchial epithelium. This study investigated the effect of increased IL33 expression on human bronchial epithelial cell (HBEC) function. METHODS: Association between IL33 SNPs (Chr9: 5,815,786-6,657,983) and asthma phenotypes (Lifelines/DAG [Dutch Asthma GWAS]/GASP [Genetics of Asthma Severity & Phenotypes] cohorts) and between SNPs and expression (lung tissue, bronchial brushes, HBECs) was done using regression modeling. Lentiviral overexpression was used to study IL33 effects on HBECs. RESULTS: We found that 161 SNPs spanning the IL33 region associated with 1 or more asthma phenotypes after correction for multiple testing. We report a main independent signal tagged by rs992969 associating with blood eosinophil levels, asthma, and eosinophilic asthma. A second, independent signal tagged by rs4008366 presented modest association with eosinophilic asthma. Neither signal associated with FEV1, FEV1/forced vital capacity, atopy, and age of asthma onset. The 2 IL33 signals are expression quantitative loci in bronchial brushes and cultured HBECs, but not in lung tissue. IL33 overexpression in vitro resulted in reduced viability and reactive oxygen species-capturing of HBECs, without influencing epithelial cell count, metabolic activity, or barrier function. CONCLUSIONS: We identify IL33 as an epithelial susceptibility gene for eosinophilia and asthma, provide mechanistic insight, and implicate targeting of the IL33 pathway specifically in eosinophilic asthma.

Multi-omics highlights ABO plasma protein as a causal risk factor for COVID-19.

SARS-CoV-2 is responsible for the coronavirus disease 2019 (COVID-19) and the current health crisis. Despite intensive research efforts, the genes and pathways that contribute to COVID-19 remain poorly understood. We, therefore, used an integrative genomics (IG) approach to identify candidate genes responsible for COVID-19 and its severity. We used Bayesian colocalization (COLOC) and summary-based Mendelian randomization to combine gene expression quantitative trait loci (eQTLs) from the Lung eQTL (n = 1,038) and eQTLGen (n = 31,784) studies with published COVID-19 genome-wide association study (GWAS) data from the COVID-19 Host Genetics Initiative. Additionally, we used COLOC to integrate plasma protein quantitative trait loci (pQTL) from the INTERVAL study (n = 3,301) with COVID-19 loci. Finally, we determined any causal associations between plasma proteins and COVID-19 using multi-variable two-sample Mendelian randomization (MR). The expression of 18 genes in lung and/or blood co-localized with COVID-19 loci. Of these, 12 genes were in suggestive loci (PGWAS < 5 × 10-05). LZTFL1, SLC6A20, ABO, IL10RB and IFNAR2 and OAS1 had been previously associated with a heightened risk of COVID-19 (PGWAS < 5 × 10-08). We identified a causal association between OAS1 and COVID-19 GWAS. Plasma ABO protein, which is associated with blood type in humans, demonstrated a significant causal relationship with COVID-19 in the MR analysis; increased plasma levels were associated with an increased risk of COVID-19 and, in particular, severe COVID-19. In summary, our study identified genes associated with COVID-19 that may be prioritized for future investigations. Importantly, this is the first study to demonstrate a causal association between plasma ABO protein and COVID-19.

Genome-Wide Association Study of Susceptibility to Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a complex lung disease characterized by scarring of the lung that is believed to result from an atypical response to injury of the epithelium. Genome-wide association studies have reported signals of association implicating multiple pathways including host defense, telomere maintenance, signaling, and cell-cell adhesion.Objectives: To improve our understanding of factors that increase IPF susceptibility by identifying previously unreported genetic associations.Methods: We conducted genome-wide analyses across three independent studies and meta-analyzed these results to generate the largest genome-wide association study of IPF to date (2,668 IPF cases and 8,591 controls). We performed replication in two independent studies (1,456 IPF cases and 11,874 controls) and functional analyses (including statistical fine-mapping, investigations into gene expression, and testing for enrichment of IPF susceptibility signals in regulatory regions) to determine putatively causal genes. Polygenic risk scores were used to assess the collective effect of variants not reported as associated with IPF.Measurements and Main Results: We identified and replicated three new genome-wide significant (P < 5 × 10-8) signals of association with IPF susceptibility (associated with altered gene expression of KIF15, MAD1L1, and DEPTOR) and confirmed associations at 11 previously reported loci. Polygenic risk score analyses showed that the combined effect of many thousands of as yet unreported IPF susceptibility variants contribute to IPF susceptibility.Conclusions: The observation that decreased DEPTOR expression associates with increased susceptibility to IPF supports recent studies demonstrating the importance of mTOR signaling in lung fibrosis. New signals of association implicating KIF15 and MAD1L1 suggest a possible role of mitotic spindle-assembly genes in IPF susceptibility.

Letermovir Resistance Analysis in a Clinical Trial of Cytomegalovirus Prophylaxis for Hematopoietic Stem Cell Transplant Recipients.

BACKGROUND: Letermovir (LET), a cytomegalovirus (CMV) deoxyribonucleic acid (DNA) terminase inhibitor, was recently approved for prophylaxis of CMV infection in adult CMV-seropositive recipients of allogeneic hematopoietic stem cell transplantation. Cytomegalovirus genotyping was performed to identify LET-resistance-associated variants (RAVs) among subjects in a Phase 3 trial. METHODS: The CMV UL56 and UL89 genes, encoding subunits of CMV DNA terminase, were sequenced from plasma collected from subjects with clinically significant CMV infection (CS-CMVi). Novel variants were evaluated by recombinant phenotyping to assess their potential to confer resistance to LET. RESULTS: Genotyping was successful for 50 of 79 LET subjects with CS-CMVi. Resistance-associated variants (encoding pUL56 V236M and C325W) were detected independently in subjects 1 and 3 who experienced CS-CMVi while receiving LET prophylaxis, and 2 other variants (encoding pUL56 E237G and R369T) were detected >3 weeks after subjects 2 and 3, respectively, had discontinued LET prophylaxis and received preemptive therapy with ganciclovir. CONCLUSIONS: The detected incidence of CMV resistance among subjects who received LET as prophylaxis in this Phase 3 trial was low. The LET RAVs that were detected mapped to the CMV UL56 gene at positions associated with reduced susceptibility to LET based on resistance selections in cell culture.

Leveraging lung tissue transcriptome to uncover candidate causal genes in COPD genetic associations.

Causal genes of chronic obstructive pulmonary disease (COPD) remain elusive. The current study aims at integrating genome-wide association studies (GWAS) and lung expression quantitative trait loci (eQTL) data to map COPD candidate causal genes and gain biological insights into the recently discovered COPD susceptibility loci. Two complementary genomic datasets on COPD were studied. First, the lung eQTL dataset which included whole-genome gene expression and genotyping data from 1038 individuals. Second, the largest COPD GWAS to date from the International COPD Genetics Consortium (ICGC) with 13 710 cases and 38 062 controls. Methods that integrated GWAS with eQTL signals including transcriptome-wide association study (TWAS), colocalization and Mendelian randomization-based (SMR) approaches were used to map causality genes, i.e. genes with the strongest evidence of being the functional effector at specific loci. These methods were applied at the genome-wide level and at COPD risk loci derived from the GWAS literature. Replication was performed using lung data from GTEx. We collated 129 non-overlapping risk loci for COPD from the GWAS literature. At the genome-wide scale, 12 new COPD candidate genes/loci were revealed and six replicated in GTEx including CAMK2A, DMPK, MYO15A, TNFRSF10A, BTN3A2 and TRBV30. In addition, we mapped candidate causal genes for 60 out of the 129 GWAS-nominated loci and 23 of them were replicated in GTEx. Mapping candidate causal genes in lung tissue represents an important contribution to the genetics of COPD, enriches our biological interpretation of GWAS findings, and brings us closer to clinical translation of genetic associations.

COPD GWAS variant at 19q13.2 in relation with DNA methylation and gene expression.

Chronic obstructive pulmonary disease (COPD) is among the major health burdens in adults. While cigarette smoking is the leading risk factor, a growing number of genetic variations have been discovered to influence disease susceptibility. Epigenetic modifications may mediate the response of the genome to smoking and regulate gene expression. Chromosome 19q13.2 region is associated with both smoking and COPD, yet its functional role is unclear. Our study aimed to determine whether rs7937 (RAB4B, EGLN2), a top genetic variant in 19q13.2 region identified in genome-wide association studies of COPD, is associated with differential DNA methylation in blood (N = 1490) and gene expression in blood (N = 721) and lungs (N = 1087). We combined genetic and epigenetic data from the Rotterdam Study (RS) to perform the epigenome-wide association analysis of rs7937. Further, we used genetic and transcriptomic data from blood (RS) and from lung tissue (Lung expression quantitative trait loci mapping study), to perform the transcriptome-wide association study of rs7937. Rs7937 was significantly (FDR < 0.05) and consistently associated with differential DNA methylation in blood at 4 CpG sites in cis, independent of smoking. One methylation site (cg11298343-EGLN2) was also associated with COPD (P = 0.001). Additionally, rs7937 was associated with gene expression levels in blood in cis (EGLN2), 42% mediated through cg11298343, and in lung tissue, in cis and trans (NUMBL, EGLN2, DNMT3A, LOC101929709 and PAK2). Our results suggest that changes of DNA methylation and gene expression may be intermediate steps between genetic variants and COPD, but further causal studies in lung tissue should confirm this hypothesis.

Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH/gL/pUL128-131 Pentameric Complex.

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigen-the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains.IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen-the pentameric complex. We further demonstrated that following vaccination of a replication-defective virus with the restored pentameric complex, rhesus macaques can develop broadly neutralizing antibodies targeting multiple immunogenic sites of the pentameric complex. Such analyses of site-specific antibody responses are imperative to our assessment of the quality of vaccine-induced immunity in clinical studies.

Genome-Wide Interaction Analysis of Air Pollution Exposure and Childhood Asthma with Functional Follow-up.

RATIONALE: The evidence supporting an association between traffic-related air pollution exposure and incident childhood asthma is inconsistent and may depend on genetic factors. OBJECTIVES: To identify gene-environment interaction effects on childhood asthma using genome-wide single-nucleotide polymorphism (SNP) data and air pollution exposure. Identified loci were further analyzed at epigenetic and transcriptomic levels. METHODS: We used land use regression models to estimate individual air pollution exposure (represented by outdoor NO2 levels) at the birth address and performed a genome-wide interaction study for doctors' diagnoses of asthma up to 8 years in three European birth cohorts (n = 1,534) with look-up for interaction in two separate North American cohorts, CHS (Children's Health Study) and CAPPS/SAGE (Canadian Asthma Primary Prevention Study/Study of Asthma, Genetics and Environment) (n = 1,602 and 186 subjects, respectively). We assessed expression quantitative trait locus effects in human lung specimens and blood, as well as associations among air pollution exposure, methylation, and transcriptomic patterns. MEASUREMENTS AND MAIN RESULTS: In the European cohorts, 186 SNPs had an interaction P < 1 × 10-4 and a look-up evaluation of these disclosed 8 SNPs in 4 loci, with an interaction P < 0.05 in the large CHS study, but not in CAPPS/SAGE. Three SNPs within adenylate cyclase 2 (ADCY2) showed the same direction of the interaction effect and were found to influence ADCY2 gene expression in peripheral blood (P = 4.50 × 10-4). One other SNP with P < 0.05 for interaction in CHS, rs686237, strongly influenced UDP-Gal:betaGlcNAc ß-1,4-galactosyltransferase, polypeptide 5 (B4GALT5) expression in lung tissue (P = 1.18 × 10-17). Air pollution exposure was associated with differential discs, large homolog 2 (DLG2) methylation and expression. CONCLUSIONS: Our results indicated that gene-environment interactions are important for asthma development and provided supportive evidence for interaction with air pollution for ADCY2, B4GALT5, and DLG2.

Genome-wide association study on the FEV1/FVC ratio in never-smokers identifies HHIP and FAM13A.

BACKGROUND: Although a striking proportion (25% to 45%) of patients with chronic obstructive pulmonary disease are never-smokers, most genetic susceptibility studies have not focused on this group exclusively. OBJECTIVE: The aim of this study was to identify common genetic variants associated with FEV1 and its ratio to forced vital capacity (FVC) in never-smokers. METHODS: Genome-wide association studies were performed in 5070 never-smokers of the identification cohort LifeLines, and results (P < 10-5) were verified by using a meta-analysis of the Vlagtwedde-Vlaardingen study and the Rotterdam Study I-III (total n = 1966). Furthermore, we aimed to assess the effects of the replicated variants in more detail by performing genetic risk score, expression quantitative trait loci, and variant*ever-smoking interaction analyses. RESULTS: We identified associations between the FEV1/FVC ratio and 5 common genetic variants in the identification cohort, and 2 of these associations were replicated. The 2 variants annotated to the genes hedgehog interacting protein (HHIP) and family with sequence similarity 13 member A (FAM13A) were shown to have an additive effect on FEV1/FVC levels in the genetic risk score analysis; were associated with gene expression of HHIP and FAM13A in lung tissue, respectively; and were genome-wide significant in a meta-analysis including both identification and 4 verification cohorts (P < 2.19 × 10-7). Finally, we did not identify significant interactions between the variants and ever smoking. Results of the FEV1 identification analysis were not replicated. CONCLUSION: The genes HHIP and FAM13A confer a risk for airway obstruction in general that is not driven exclusively by cigarette smoking, which is the main risk factor for chronic obstructive pulmonary disease.

Integrative Genomics of Emphysema-Associated Genes Reveals Potential Disease Biomarkers.

Chronic obstructive pulmonary disease is the third leading cause of death worldwide. Gene expression profiling across multiple regions of the same lung identified genes significantly related to emphysema. We sought to determine whether the lung and epithelial expression of 127 emphysema-related genes was also related to lung function in independent cohorts, and whether any of these genes could be used as biomarkers in the peripheral blood of patients with chronic obstructive pulmonary disease. To that end, we examined whether the expression levels of these genes were under genetic control in lung tissue (n = 1,111). We then determined whether the mRNA levels of these genes in lung tissue (n = 727), small airway epithelial cells (n = 238), and peripheral blood (n = 620) were significantly related to lung function measurements. The expression of 63 of the 127 genes (50%) was under genetic control in lung tissue. The lung and epithelial mRNA expression of a subset of the emphysema-associated genes, including ASRGL1, LPHN2, and EDNRB, was strongly associated with lung function. In peripheral blood, the expression of 40 genes was significantly associated with lung function. Twenty-nine of these genes (73%) were also associated with lung function in lung tissue, but with the opposite direction of effect for 24 of the 29 genes, including those involved in hypoxia and B cell-related responses. The integrative genomics approach uncovered a significant overlap of emphysema genes associations with lung function between lung and blood with opposite directions between the two. These results support the use of peripheral blood to detect disease biomarkers.

Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons.

Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC50) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n = 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137- and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n = 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir. The activity profiles of grazoprevir and elbasvir supported the testing of the direct-acting antivirals in clinical studies.

Lung tissue gene-expression signature for the ageing lung in COPD.

INTRODUCTION: COPD is a chronic, progressive, inflammatory disease of the lungs and the third leading cause of death worldwide. The current knowledge of the pathophysiology of COPD is limited and novel insights in underlying disease mechanisms are urgently needed. Since there are clear parallels between ageing and COPD, we investigated genes underlying lung ageing in general and abnormal lung ageing in COPD. METHODS: Whole genome mRNA profiling was performed on lung tissue samples (n=1197) and differential gene expression with increasing age was analysed using an adjusted linear regression model. Subsequent pathway analysis was performed using GeneNetwork and the gene-expression signature was compared with lung ageing in the Genotype-Tissue Expression (GTEx) project. In a subset of patients with COPD (n=311) and non-COPD controls (n=270), we performed an interaction analysis between age and COPD to identify genes differentially expressed with age in COPD compared with controls, followed by gene set enrichment pathway analysis. RESULTS: We identified a strong gene-expression signature for lung ageing with 3509 differentially expressed genes, of which 33.5% were found nominal significant in the GTEx project. Interestingly, we found EDA2R as a strong candidate gene for lung ageing. The age*COPD interaction analysis revealed 69 genes significantly differentially expressed with age between COPD and controls. CONCLUSIONS: Our study indicates that processes related to lung development, cell-cell contacts, calcium signalling and immune responses are involved in lung ageing in general. Pathways related to extracellular matrix, mammalian target of rapamycin signalling, splicing of introns and exons and the ribosome complex are proposed to be involved in abnormal lung ageing in COPD.

Surfactant protein D is a causal risk factor for COPD: results of Mendelian randomisation.

Surfactant protein D (SP-D) is produced primarily in the lung and is involved in regulating pulmonary surfactants, lipid homeostasis and innate immunity. Circulating SP-D levels in blood are associated with chronic obstructive pulmonary disease (COPD), although causality remains elusive.In 4061 subjects with COPD, we identified genetic variants associated with serum SP-D levels. We then determined whether these variants affected lung tissue gene expression in 1037 individuals. A Mendelian randomisation framework was then applied, whereby serum SP-D-associated variants were tested for association with COPD risk in 11 157 cases and 36 699 controls and with 11 years decline of lung function in the 4061 individuals.Three regions on chromosomes 6 (human leukocyte antigen region), 10 (SFTPD gene) and 16 (ATP2C2 gene) were associated with serum SP-D levels at genome-wide significance. In Mendelian randomisation analyses, variants associated with increased serum SP-D levels decreased the risk of COPD (estimate -0.19, p=6.46×10-03) and slowed the lung function decline (estimate=0.0038, p=7.68×10-3).Leveraging genetic variation effect on protein, lung gene expression and disease phenotypes provided novel insights into SP-D biology and established a causal link between increased SP-D levels and protection against COPD risk and progression. SP-D represents a very promising biomarker and therapeutic target for COPD.

Sulfatase modifying factor 1 (SUMF1) is associated with Chronic Obstructive Pulmonary Disease.

BACKGROUND: It has been observed that mice lacking the sulfatase modifying factor (Sumf1) developed an emphysema-like phenotype. However, it is unknown if SUMF1 may play a role in Chronic Obstructive Pulmonary Disease (COPD) in humans. The aim was to investigate if the expression and genetic regulation of SUMF1 differs between smokers with and without COPD. METHODS: SUMF1 mRNA was investigated in sputum cells and whole blood from controls and COPD patients (all current or former smokers). Expression quantitative trait loci (eQTL) analysis was used to investigate if single nucleotide polymorphisms (SNPs) in SUMF1 were significantly associated with SUMF1 expression. The association of SUMF1 SNPs with COPD was examined in a population based cohort, Lifelines. SUMF1 mRNA from sputum cells, lung tissue, and lung fibroblasts, as well as lung function parameters, were investigated in relation to genotype. RESULTS: Certain splice variants of SUMF1 showed a relatively high expression in lung tissue compared to many other tissues. SUMF1 Splice variant 2 and 3 showed lower levels in sputum cells from COPD patients as compared to controls. Twelve SNPs were found significant by eQTL analysis and overlapped with the array used for genotyping of Lifelines. We found alterations in mRNA expression in sputum cells and lung fibroblasts associated with SNP rs11915920 (top hit in eQTL), which validated the results of the lung tissue eQTL analysis. Of the twelve SNPs, two SNPs, rs793391 and rs308739, were found to be associated with COPD in Lifelines. The SNP rs793391 was also confirmed to be associated with lung function changes. CONCLUSIONS: We show that SUMF1 expression is affected in COPD patients compared to controls, and that SNPs in SUMF1 are associated with an increased risk of COPD. Certain COPD-associated SNPs have effects on either SUMF1 gene expression or on lung function. Collectively, this study shows that SUMF1 is associated with an increased risk of developing COPD.

Elbasvir-Grazoprevir to Treat Hepatitis C Virus Infection in Persons Receiving Opioid Agonist Therapy: A Randomized Trial.

Background: Hepatitis C virus (HCV) infection is common in persons who inject drugs (PWID). Objective: To evaluate elbasvir-grazoprevir in treating HCV infection in PWID. Design: Randomized, placebo-controlled, double-blind trial. (ClinicalTrials.gov: NCT02105688). Setting: Australia, Canada, France, Germany, Israel, the Netherlands, New Zealand, Norway, Spain, Taiwan, the United Kingdom, and the United States. Patients: 301 treatment-naive patients with chronic HCV genotype 1, 4, or 6 infection who were at least 80% adherent to visits for opioid agonist therapy (OAT). Intervention: The immediate-treatment group (ITG) received elbasvir-grazoprevir for 12 weeks; the deferred-treatment group (DTG) received placebo for 12 weeks, no treatment for 4 weeks, then open-label elbasvir-grazoprevir for 12 weeks. Measurements: The primary outcome was sustained virologic response at 12 weeks (SVR12), evaluated separately in the ITG and DTG. Other outcomes included SVR24, viral recurrence or reinfection, and adverse events. Results: The SVR12 was 91.5% (95% CI, 86.8% to 95.0%) in the ITG and 89.5% (95% CI, 81.5% to 94.8%) in the active phase of the DTG. Drug use at baseline and during treatment did not affect SVR12 or adherence to HCV therapy. Among 18 patients with posttreatment viral recurrence through 24-week follow-up, 6 had probable reinfection. If the probable reinfections were assumed to be responses, SVR12 was 94.0% (CI, 89.8% to 96.9%) in the ITG. One patient in the ITG (1 of 201) and 1 in the placebo-phase DTG (1 of 100) discontinued treatment because of an adverse event. Limitation: These findings may not be generalizable to PWID who are not receiving OAT, nor do they apply to persons with genotype 3 infection, a common strain in PWID. Conclusion: Patients with HCV infection who were receiving OAT and treated with elbasvir-grazoprevir had high rates of SVR12, regardless of ongoing drug use. These results support the removal of drug use as a barrier to interferon-free HCV treatment for patients receiving OAT. Primary Funding Source: Merck & Co.

Whole exome re-sequencing implicates CCDC38 and cilia structure and function in resistance to smoking related airflow obstruction.

Chronic obstructive pulmonary disease (COPD) is a leading cause of global morbidity and mortality and, whilst smoking remains the single most important risk factor, COPD risk is heritable. Of 26 independent genomic regions showing association with lung function in genome-wide association studies, eleven have been reported to show association with airflow obstruction. Although the main risk factor for COPD is smoking, some individuals are observed to have a high forced expired volume in 1 second (FEV1) despite many years of heavy smoking. We hypothesised that these "resistant smokers" may harbour variants which protect against lung function decline caused by smoking and provide insight into the genetic determinants of lung health. We undertook whole exome re-sequencing of 100 heavy smokers who had healthy lung function given their age, sex, height and smoking history and applied three complementary approaches to explore the genetic architecture of smoking resistance. Firstly, we identified novel functional variants in the "resistant smokers" and looked for enrichment of these novel variants within biological pathways. Secondly, we undertook association testing of all exonic variants individually with two independent control sets. Thirdly, we undertook gene-based association testing of all exonic variants. Our strongest signal of association with smoking resistance for a non-synonymous SNP was for rs10859974 (P = 2.34 × 10(-4)) in CCDC38, a gene which has previously been reported to show association with FEV1/FVC, and we demonstrate moderate expression of CCDC38 in bronchial epithelial cells. We identified an enrichment of novel putatively functional variants in genes related to cilia structure and function in resistant smokers. Ciliary function abnormalities are known to be associated with both smoking and reduced mucociliary clearance in patients with COPD. We suggest that genetic influences on the development or function of cilia in the bronchial epithelium may affect growth of cilia or the extent of damage caused by tobacco smoke.

A pro-inflammatory role for the Frizzled-8 receptor in chronic bronchitis.

RATIONALE: We have previously shown increased expression of the Frizzled-8 receptor of the Wingless/integrase-1 (WNT) signalling pathway in COPD. Here, we investigated if the Frizzled-8 receptor has a functional role in airway inflammation associated with chronic bronchitis. METHODS: Acute cigarette-smoke-induced airway inflammation was studied in wild-type and Frizzled-8-deficient mice. Genetic association studies and lung expression quantitative trait loci (eQTL) analyses for Frizzled-8 were performed to evaluate polymorphisms in FZD8 and their relationship to tissue expression in chronic bronchitis. Primary human lung fibroblasts and primary human airway epithelial cells were used for in vitro studies. RESULTS: Cigarette-smoke-exposure induced airway inflammation in wild-type mice, which was prevented in Frizzled-8-deficient mice, suggesting a crucial role for Frizzled-8 in airway inflammation. Furthermore, we found a significant genetic association (p=0.009) between single nucleotide polymorphism (SNP) rs663700 in the FZD8 region and chronic mucus hypersecretion, a characteristic of chronic bronchitis, in a large cohort of smoking individuals. We found SNP rs663700 to be a cis-eQTL regulating Frizzled-8 expression in lung tissue. Functional data link mesenchymal Frizzled-8 expression to inflammation as its expression in COPD-derived lung fibroblasts was regulated by pro-inflammatory cytokines in a genotype-dependent manner. Moreover, Frizzled-8 regulates inflammatory cytokine secretion from human lung fibroblasts, which in turn promoted MUC5AC expression by differentiated human airway epithelium. CONCLUSIONS: These findings indicate an important pro-inflammatory role for Frizzled-8 and suggest that its expression is related to chronic bronchitis. Furthermore, our findings indicate an unexpected role for fibroblasts in regulating airway inflammation in COPD.