ZNF768 links oncogenic RAS to cellular senescence.

RAS proteins are GTPases that lie upstream of a signaling network impacting cell fate determination. How cells integrate RAS activity to balance proliferation and cellular senescence is still incompletely characterized. Here, we identify ZNF768 as a phosphoprotein destabilized upon RAS activation. We report that ZNF768 depletion impairs proliferation and induces senescence by modulating the expression of key cell cycle effectors and established p53 targets. ZNF768 levels decrease in response to replicative-, stress- and oncogene-induced senescence. Interestingly, ZNF768 overexpression contributes to bypass RAS-induced senescence by repressing the p53 pathway. Furthermore, we show that ZNF768 interacts with and represses p53 phosphorylation and activity. Cancer genomics and immunohistochemical analyses reveal that ZNF768 is often amplified and/or overexpressed in tumors, suggesting that cells could use ZNF768 to bypass senescence, sustain proliferation and promote malignant transformation. Thus, we identify ZNF768 as a protein linking oncogenic signaling to the control of cell fate decision and proliferation.

Prioritization of candidate causal genes for asthma in susceptibility loci derived from UK Biobank.

To identify candidate causal genes of asthma, we performed a genome-wide association study (GWAS) in UK Biobank on a broad asthma definition (n = 56,167 asthma cases and 352,255 controls). We then carried out functional mapping through transcriptome-wide association studies (TWAS) and Mendelian randomization in lung (n = 1,038) and blood (n = 31,684) tissues. The GWAS reveals 72 asthma-associated loci from 116 independent significant variants (PGWAS < 5.0E-8). The most significant lung TWAS gene on 17q12-q21 is GSDMB (PTWAS = 1.42E-54). Other TWAS genes include TSLP on 5q22, RERE on 1p36, CLEC16A on 16p13, and IL4R on 16p12, which all replicated in GTEx lung (n = 515). We demonstrate that the largest fold enrichment of regulatory and functional annotations among asthma-associated variants is in the blood. We map 485 blood eQTL-regulated genes associated with asthma and 50 of them are causal by Mendelian randomization. Prioritization of druggable genes reveals known (IL4R, TSLP, IL6, TNFSF4) and potentially new therapeutic targets for asthma.

Linoleic acid supplementation of cell culture media influences the phospholipid and lipid profiles of human reconstructed adipose tissue.

Reconstructed human adipose tissues represent novel tools available to perform in vitro pharmaco-toxicological studies. We used adipose-derived human stromal/stem cells to reconstruct, using tissue engineering techniques, such an adipose tridimensional model. To determine to what extent the in vitro model is representative of its native counterpart, adipogenic differentiation, triglycerides accumulation and phospholipids profiles were analysed. Ingenuity Pathway Analysis software revealed pathways enriched with differentially-expressed genes between native and reconstructed human adipose tissues. Interestingly, genes related to fatty acid metabolism were downregulated in vitro, which could be explained in part by the insufficient amount of essential fatty acids provided by the fetal calf serum used for the culture. Indeed, the lipid profile of the reconstructed human adipose tissues indicated a particular lack of linoleic acid, which could interfere with physiological cell processes such as membrane trafficking, signaling and inflammatory responses. Supplementation in the culture medium was able to influence the lipid profile of the reconstructed human adipose tissues. This study demonstrates the possibility to directly modulate the phospholipid profile of reconstructed human adipose tissues. This reinforces its use as a relevant physiological or pathological model for further pharmacological and metabolic studies of human adipose tissue functions.

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.

Deleterious variants in DCHS1 are prevalent in sporadic cases of mitral valve prolapse.

BACKGROUND: A recent study identified DCHS1 as a causal gene for mitral valve prolapse. The goal of this study is to investigate the presence and frequency of known and novel variants in this gene in 100 asymptomatic patients with moderate to severe organic mitral regurgitation. METHODS: DNA sequencing assays were developed for two previously identified functional missense variants, namely p.R2330C and p.R2513H, and all 21 exons of DCHS1. Pathogenicity of variants was evaluated in silico. RESULTS: p.R2330C and p.R2513H were not identified in this cohort. Sequencing all coding regions revealed eight missense variants including six considered deleterious. This includes one novel variant (p.A2464P) and two rare variants (p.R2770Q and p.R2462Q). These variants are predicted to be deleterious with combined annotation-dependent depletion (CADD) scores greater than 25, which are in the same range as p.R2330C (CADD = 28.0) and p.R2513H (CADD = 24.3). More globally, 24 of 100 cases were carriers of at least one in silico-predicted deleterious missense variant in DCHS1, suggesting that this single gene may account for a substantial portion of cases. CONCLUSION: This study reveals an important contribution of germline variants in DCHS1 in unrelated patients with mitral valve prolapse and supports genetic testing of this gene to screen individuals at risk.

Exposure to electronic cigarette vapors affects pulmonary and systemic expression of circadian molecular clock genes.

E-cigarette use has exploded in the past years, especially among young adults and smokers desiring to quit. While concerns are mostly based on the presence of nicotine and flavors, pulmonary effects of propylene glycol and glycerol inhalation, the main solvents of e-liquid have not been thoroughly investigated. In this preclinical study, mice were exposed 2 h daily for up to 8 weeks to vapors of propylene glycol and/or glycerol generated by an e-cigarette. Lung transcriptome analysis revealed it affected the expression level of genes of the circadian molecular clock, despite causing no inflammatory response. Periodical sacrifices showed that the rhythmicity of these regulatory genes was indeed altered in the lungs, but also in the liver, kidney, skeletal muscle, and brain. E-cigarette exposure also altered the expression of rhythmic genes (i.e., hspa1a and hspa1b), suggesting that alterations to the 'clock genes' could translate into systemic biological alterations. This study reveals that the major solvents used in e-cigarettes propylene glycol and glycerol, not nicotine or flavors, have unsuspected effects on gene expression of the molecular clock that are to be taken seriously, especially considering the fundamental role of the circadian rhythm in health and disease.

Interplay between cigarette smoking and pulmonary reverse lipid transport.

Reverse lipid transport is critical to maintain homeostasis. Smoking causes lipid accumulation in macrophages, therefore suggesting suboptimal reverse lipid transport mechanisms. In this study, we investigated the interplay between smoking and reverse lipid transport and the consequences on smoking-induced lung and peripheral alterations.To investigate the relationship between smoking and reverse lipid transport, we used a clinical lung gene expression dataset and a mouse model of cigarette smoke exposure. We also used ApoA-1-/- mice, with reduced reverse lipid transport capacity, and a recombinant ApoA-1 Milano/phospholipid complex (MDCO-216) to boost reverse lipid transport. Cellular and functional analyses were performed on the lungs and impact on body composition was also assessed.Smoking affects pulmonary expression of abca1, abcg1, apoe and scarb1 in both mice and humans, key genes involved in reverse lipid transport. In mice, the capacity of bronchoalveolar lavage fluid and serum to stimulate cholesterol efflux in macrophages was increased after a single exposure to cigarette smoke. ApoA-1-/- mice showed increased lung neutrophilia, larger macrophages and greater loss in lean mass in response to smoking, whereas treatment with MDCO-216 reduced the size of macrophages and increased the lean mass of mice exposed to cigarette smoke.Altogether, this study shows a functional interaction between smoking and reverse lipid transport, and opens new avenues for better understanding the link between metabolic and pulmonary diseases related to smoking.

Identification of Susceptibility Genes of Adult Asthma in French Canadian Women.

Susceptibility genes of asthma may be more successfully identified by studying subgroups of phenotypically similar asthma patients. This study aims to identify single nucleotide polymorphisms (SNPs) associated with asthma in French Canadian adult women. A pooling-based genome-wide association study was performed in 240 allergic asthmatic and 120 allergic nonasthmatic women. The top associated SNPs were selected for individual genotyping in an extended cohort of 349 asthmatic and 261 nonasthmatic women. The functional impact of asthma-associated SNPs was investigated in a lung expression quantitative trait loci (eQTL) mapping study (n = 1035). Twenty-one of the 38 SNPs tested by individual genotyping showed P values lower than 0.05 for association with asthma. Cis-eQTL analyses supported the functional contribution of rs17801353 associated with C3AR1 (P = 7.90E - 10). The asthma risk allele for rs17801353 is associated with higher mRNA expression levels of C3AR1 in lung tissue. In silico functional characterization of the asthma-associated SNPs also supported the contribution of C3AR1 and additional genes including SYNE1, LINGO2, and IFNG-AS1. This pooling-based GWAS in French Canadian adult women followed by lung eQTL mapping suggested C3AR1 as a functional locus associated with asthma. Additional susceptibility genes were suggested in this homogenous subgroup of asthma patients.

Asthma susceptibility variants are more strongly associated with clinically similar subgroups.

OBJECTIVE: Genome-wide association studies (GWAS) identified single nucleotide polymorphisms (SNPs) reproducibly associated with asthma. This study evaluated whether GWAS-nominated SNPs are more strongly associated with asthma patients sharing the same clinical characteristics in order to refine the role of recently identified genes. METHODS: Analyses were performed in unrelated French Canadian subjects (566 cases and 416 controls) with data collected on lung function, blood cell counts, atopy, disease history and medication. Previously defined asthma subgroups were used for analysis: 1) older patients with low atopy and low lung function, 2) high atopy, 3) young non-smoking women and 4) high smoking history. Allele frequencies of 68 GWAS-nominated SNPs were compared between controls and cases or controls and subgroups of cases defined by cluster analysis. RESULTS: Twelve GWAS-nominated SNPs demonstrated evidence of replication (p value < 0.05) for association with asthma. In phenotypically similar asthma patients, rs10197862, located in IL1RL1/IL18R1, was the most strongly associated SNP with the high atopy subgroup (p = 0.0009). SNPs located at the IL33 and the STARD3/PGAP3 loci were also associated with the high atopy subgroup. Two SNPs, rs1544791 (PDE4D) and rs3806932 (TSLP), were more strongly associated with the high smoking history subgroup than with asthma or any other subgroups. All 10 SNPs that replicated for asthma per se and within subgroups had lower p values in subgroups. Moreover, 12 SNPs were only replicated in a subgroup. CONCLUSION: This study shows that the majority of GWAS-nominated SNPs are more strongly associated with homogeneous subgroups of asthma than broadly defined asthma.

Role of BAFF in pulmonary autoantibody responses induced by chronic cigarette smoke exposure in mice.

Emerging evidence suggests that autoimmune processes are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). In this study, we assessed the expression of B-cell activating factor (BAFF) in smokers, and investigated the functional importance of BAFF in the induction and maintenance of cigarette smoke-induced pulmonary antinuclear antibodies (ANA) and tertiary lymphoid tissues (TLTs) using a preclinical mouse model. We observed that BAFF levels were elevated in smokers and mice exposed to cigarette smoke. In mice, BAFF expression was rapidly induced in the lungs following 4 days of cigarette smoke exposure and remained elevated following 8 and 24 weeks of exposure. Alveolar macrophages were the major source of BAFF Blockade of BAFF using a BAFF receptor-Fc (BAFFR-Fc) construct prevented pulmonary ANA and TLT formation when delivered concurrent with cigarette smoke exposure. Under these conditions, no impact on lung inflammation was observed. However, administration of BAFFR-Fc following smoking cessation markedly reduced the number of TLTs and ANA levels and, of note, reduced pulmonary neutrophilia. Altogether, this study shows for the first time a central role of BAFF in the induction and maintenance of cigarette smoke-induced pulmonary ANA and suggests that BAFF blockade following smoking cessation could have beneficial effects on persistent inflammatory processes.In this study, we assessed the expression of B-cell activating factor (BAFF) in smokers, and investigated the functional importance of BAFF in the induction and maintenance of cigarette smoke-induced pulmonary antinuclear antibodies (ANA) and tertiary lymphoid tissues (TLTs) using a preclinical mouse model. Data presented show that BAFF plays a central role in the induction and maintenance of cigarette smoke-induced pulmonary ANA and suggest a therapeutic potential for BAFF blockade in limiting autoimmune processes associated with smoking.

Altered intestinal functions and increased local inflammation in insulin-resistant obese subjects: a gene-expression profile analysis.

BACKGROUND: Metabolic alterations relevant to postprandial dyslipidemia were previously identified in the intestine of obese insulin-resistant subjects. The aim of the study was to identify the genes deregulated by systemic insulin resistance in the intestine of severely obese subjects. METHODS: Transcripts from duodenal samples of insulin-sensitive (HOMA-IR < 3, n = 9) and insulin-resistant (HOMA-IR > 7, n = 9) obese subjects were assayed by microarray (Illumina HumanHT-12). RESULTS: A total of 195 annotated genes were identified as differentially expressed between these two groups (Fold change > 1.2). Of these genes, 36 were found to be directly involved in known intestinal functions, including digestion, extracellular matrix, endocrine system, immunity and cholesterol metabolism. Interestingly, all differentially expressed genes (n = 8) implicated in inflammation and oxidative stress were found to be upregulated in the intestine of insulin-resistant compared to insulin-sensitive subjects. Metabolic pathway analysis revealed that several signaling pathways involved in immunity and inflammation were significantly enriched in differently expressed genes and were predicted to be activated in the intestine of insulin-resistant subjects. Using stringent criteria (Fold change > 1.5; FDR < 0.05), three genes were found to be significantly and differently expressed in the intestine of insulin-resistant compared to insulin-sensitive subjects: the transcripts of the insulinotropic glucose-dependant peptide (GIP) and of the ß-microseminoprotein (MSMB) were significantly reduced, but that of the humanin like-1 (MTRNR2L1) was significantly increased. CONCLUSION: These results underline that systemic insulin resistance is associated with remodeling of key intestinal functions. Moreover, these data indicate that small intestine metabolic dysfunction is accompanied with a local amplification of low-grade inflammatory process implicating several pathways. Genes identified in this study are potentially triggered throughout the development of intestinal metabolic abnormalities, which could contribute to dyslipidemia, a component of metabolic syndrome and diabetes.

Polymorphisms associated with expression of BPIFA1/BPIFB1 and lung disease severity in cystic fibrosis.

BPI fold containing family A, member 1 (BPIFA1) and BPIFB1 are putative innate immune molecules expressed in the upper airways. Because of their hypothesized roles in airway defense, these molecules may contribute to lung disease severity in cystic fibrosis (CF). We interrogated BPIFA1/BPIFB1 single-nucleotide polymorphisms in data from an association study of CF modifier genes and found an association of the G allele of rs1078761 with increased lung disease severity (P = 2.71 × 10(-4)). We hypothesized that the G allele of rs1078761 is associated with decreased expression of BPIFA1 and/or BPIFB1. Genome-wide lung gene expression and genotyping data from 1,111 individuals with lung disease, including 51 patients with CF, were tested for associations between genotype and BPIFA1 and BPIFB1 gene expression levels. Findings were validated by quantitative PCR in a subset of 77 individuals. Western blotting was used to measure BPIFA1 and BPIFB1 protein levels in 93 lung and 101 saliva samples. The G allele of rs1078761 was significantly associated with decreased mRNA levels of BPIFA1 (P = 4.08 × 10(-15)) and BPIFB1 (P = 0.0314). These findings were confirmed with quantitative PCR and Western blotting. We conclude that the G allele of rs1078761 may be detrimental to lung function in CF owing to decreased levels of BPIFA1 and BPIFB1.

Future clinical implications emerging from recent genome-wide expression studies in asthma.

Host susceptibility to environmental triggers is the most likely explanation for the development of asthma. Quantifying gene expression levels in disease-relevant tissues and cell types using fast evolving genomic technologies have generated new hypotheses about the pathogenesis of asthma and identified new therapeutic targets to treat asthma and asthma-exacerbations. New biomarkers and distinct transcriptomic phenotypes in blood, sputum and other tissues were also identified and proved effective to refine asthma classification and guide targeted therapies. The wealth of information provided by transcriptomic studies in asthma is yet to be fully exploited, but discoveries in this field may soon be implemented in clinical settings to improve diagnosis and treatment of patients afflicted with this common disease.

Impact of cigarette smoke on the human and mouse lungs: a gene-expression comparison study.

Cigarette smoke is well known for its adverse effects on human health, especially on the lungs. Basic research is essential to identify the mechanisms involved in the development of cigarette smoke-related diseases, but translation of new findings from pre-clinical models to the clinic remains difficult. In the present study, we aimed at comparing the gene expression signature between the lungs of human smokers and mice exposed to cigarette smoke to identify the similarities and differences. Using human and mouse whole-genome gene expression arrays, changes in gene expression, signaling pathways and biological functions were assessed. We found that genes significantly modulated by cigarette smoke in humans were enriched for genes modulated by cigarette smoke in mice, suggesting a similar response of both species. Sixteen smoking-induced genes were in common between humans and mice including six newly reported to be modulated by cigarette smoke. In addition, we identified a new conserved pulmonary response to cigarette smoke in the induction of phospholipid metabolism/degradation pathways. Finally, the majority of biological functions modulated by cigarette smoke in humans were also affected in mice. Altogether, the present study provides information on similarities and differences in lung gene expression response to cigarette smoke that exist between human and mouse. Our results foster the idea that animal models should be used to study the involvement of pathways rather than single genes in human diseases.