Rationale: Chronic obstructive pulmonary disease (COPD) is associated with local (lung) and systemic (blood) inflammation and manifestations. DNA methylation is an important regulator of gene transcription, and global and specific gene methylation marks may vary with cigarette smoke exposure.
Objectives: To perform a comprehensive assessment of methylation marks in DNA from subjects well phenotyped for nonneoplastic lung disease.
Methods: We conducted array-based methylation screens, using a test-replication approach, in two family-based cohorts (n = 1,085 and 369 subjects).
Measurements and Main Results: We observed 349 CpG sites significantly associated with the presence and severity of COPD in both cohorts. Seventy percent of the associated CpG sites were outside of CpG islands, with the majority of CpG sites relatively hypomethylated. Gene ontology analysis based on these 349 CpGs (330 genes) suggested the involvement of a number of genes responsible for immune and inflammatory system pathways, responses to stress and external stimuli, as well as wound healing and coagulation cascades. Interestingly, our observations include significant, replicable associations between SERPINA1 hypomethylation and COPD and lower average lung function phenotypes (combined P values: COPD, 1.5 × 10−23; FEV1/FVC, 1.5 × 10−35; FEV1, 2.2 × 10−40).
Conclusions: Genetic and epigenetic pathways may both contribute to COPD. Many of the top associations between COPD and DNA methylation occur in biologically plausible pathways. This large-scale analysis suggests that DNA methylation may be a biomarker of COPD and may highlight new pathways of COPD pathogenesis.
chronic obstructive pulmonary disease; epigenetics; DNA methylation; smoking
The genetic risk factors for chronic obstructive pulmonary disease (COPD) are still largely unknown. To date, genome-wide association studies (GWASs) of limited size have identified several novel risk loci for COPD at CHRNA3/CHRNA5/IREB2, HHIP and FAM13A; additional loci may be identified through larger studies. We performed a GWAS using a total of 3499 cases and 1922 control subjects from four cohorts: the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE); the Normative Aging Study (NAS) and National Emphysema Treatment Trial (NETT); Bergen, Norway (GenKOLS); and the COPDGene study. Genotyping was performed on Illumina platforms with additional markers imputed using 1000 Genomes data; results were summarized using fixed-effect meta-analysis. We identified a new genome-wide significant locus on chromosome 19q13 (rs7937, OR = 0.74, P = 2.9 × 10−9). Genotyping this single nucleotide polymorphism (SNP) and another nearby SNP in linkage disequilibrium (rs2604894) in 2859 subjects from the family-based International COPD Genetics Network study (ICGN) demonstrated supportive evidence for association for COPD (P = 0.28 and 0.11 for rs7937 and rs2604894), pre-bronchodilator FEV1 (P = 0.08 and 0.04) and severe (GOLD 3&4) COPD (P = 0.09 and 0.017). This region includes RAB4B, EGLN2, MIA and CYP2A6, and has previously been identified in association with cigarette smoking behavior.
Two recent metaanalyses of genome-wide association studies conducted by the CHARGE and SpiroMeta consortia identified novel loci yielding evidence of association at or near genome-wide significance (GWS) with FEV1 and FEV1/FVC. We hypothesized that a subset of these markers would also be associated with chronic obstructive pulmonary disease (COPD) susceptibility. Thirty-two single-nucleotide polymorphisms (SNPs) in or near 17 genes in 11 previously identified GWS spirometric genomic regions were tested for association with COPD status in four COPD case-control study samples (NETT/NAS, the Norway case-control study, ECLIPSE, and the first 1,000 subjects in COPDGene; total sample size, 3,456 cases and 1,906 controls). In addition to testing the 32 spirometric GWS SNPs, we tested a dense panel of imputed HapMap2 SNP markers from the 17 genes located near the 32 GWS SNPs and in a set of 21 well studied COPD candidate genes. Of the previously identified GWS spirometric genomic regions, three loci harbored SNPs associated with COPD susceptibility at a 5% false discovery rate: the 4q24 locus including FLJ20184/INTS12/GSTCD/NPNT, the 6p21 locus including AGER and PPT2, and the 5q33 locus including ADAM19. In conclusion, markers previously associated at or near GWS with spirometric measures were tested for association with COPD status in data from four COPD case-control studies, and three loci showed evidence of association with COPD susceptibility at a 5% false discovery rate.
Traditional genome-wide association studies (GWAS) of large cohort 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.
We hypothesize that by applying unbiased weights derived from unique populations we can identify additional COPD susceptibility loci.
We performed a homozygosity haplotype analysis on a group of subjects with and without COPD to identify regions of conserved homozygosity (RCHH). Weights were constructed based on the frequency of these RCHH in case vs. controls, and used to adjust the P values from a large collaborative GWAS of COPD.
We identified 2,318 regions of conserved homozygosity, of which 576 were significantly (P < .05) overrepresented in cases. After applying the weights constructed from these regions to a collaborative GWAS of COPD, we identified two single nucleotide polymorphisms in a novel gene (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-07 and 2.8E-06 respectively). In another independent population, increased lung tissue FGF7 expression was associated with worse measures of lung function.
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.
Disease causing bacteria often manipulate host cells in a way that facilitates the infectious process. Many pathogenic gram-negative bacteria accomplish this by using type III secretion systems. In these complex secretion pathways, bacterial chaperones direct effector proteins to a needle-like secretion apparatus, which then delivers the effector protein into the host cell cytosol. The effector protein ExoU and its chaperone SpcU are components of the Pseudomonas aeruginosa type III secretion system. Secretion of ExoU has been associated with more severe infections in both humans and animal models. Here we describe the 1.92 Å X-ray structure of the ExoU–SpcU complex, a full-length type III effector in complex with its full-length cognate chaperone. Our crystallographic data allow a better understanding of the mechanism by which ExoU kills host cells and provides a foundation for future studies aimed at designing inhibitors of this potent toxin.
Cigarette smoking is a major risk factor for COPD and COPD severity. Previous genome-wide association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) associated with the number of cigarettes smoked per day (CPD) and a Dopamine Beta-Hydroxylase (DBH) locus associated with smoking cessation in multiple populations.
To identify SNPs associated with lifetime average and current CPD, age at smoking initiation, and smoking cessation in COPD subjects.
GWAS were conducted in 4 independent cohorts encompassing 3,441 ever-smoking COPD subjects (GOLD stage II or higher). Untyped SNPs were imputed using HapMap (phase II) panel. Results from all cohorts were meta-analyzed.
Several SNPs near the HLA region on chromosome 6p21 and in an intergenic region on chromosome 2q21 showed associations with age at smoking initiation, both with the lowest p=2×10−7. No SNPs were associated with lifetime average CPD, current CPD or smoking cessation with p<10−6. Nominally significant associations with candidate SNPs within alpha-nicotinic acetylcholine receptors 3/5 (CHRNA3/CHRNA5; e.g. p=0.00011 for SNP rs1051730) and Cytochrome P450 2A6 (CYP2A6; e.g. p=2.78×10−5 for a nonsynonymous SNP rs1801272) regions were observed for lifetime average CPD, however only CYP2A6 showed evidence of significant association with current CPD. A candidate SNP (rs3025343) in the DBH was significantly (p=0.015) associated with smoking cessation.
We identified two candidate regions associated with age at smoking initiation in COPD subjects. Associations of CHRNA3/CHRNA5 and CYP2A6 loci with CPD and DBH with smoking cessation are also likely of importance in the smoking behaviors of COPD patients.
Chronic Obstructive Pulmonary Disease (COPD); Genome Wide Association study (GWAS); smoking behaviors; Single Nucleotide Polymorphism (SNP)
The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS.
Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holo form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpSSA), Vibrio cholerae (AcpSVC) and Bacillus anthracis (AcpSBA) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpSBA is emphasized because of the two 3′,5′-adenosine diphosphate (3′,5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′,5′-ADP is bound as the 3′,5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′,5′-ADP–AcpS binary complexes. The position of the second 3′,5′-ADP has never been described before. It is in close proximity to the first 3′,5′-ADP and the ACP-binding site. The coordination of two ADPs in AcpSBA may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.
acyl-carrier-protein synthase; acyl carrier protein; type II fatty-acid synthesis; inhibition; 3′,5′-adenosine diphosphate; coenzyme A
Cachexia, whether assessed by body mass index (BMI) or fat-free mass index (FFMI), affects a significant proportion of patients with chronic obstructive pulmonary disease (COPD), and is an independent risk factor for increased mortality, increased emphysema, and more severe airflow obstruction. The variable development of cachexia among patients with COPD suggests a role for genetic susceptibility. The objective of the present study was to determine genetic susceptibility loci involved in the development of low BMI and FFMI in subjects with COPD. A genome-wide association study (GWAS) of BMI was conducted in three independent cohorts of European descent with Global Initiative for Chronic Obstructive Lung Disease stage II or higher COPD: Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-Points (ECLIPSE; n = 1,734); Norway-Bergen cohort (n = 851); and a subset of subjects from the National Emphysema Treatment Trial (NETT; n = 365). A genome-wide association of FFMI was conducted in two of the cohorts (ECLIPSE and Norway). In the combined analyses, a significant association was found between rs8050136, located in the first intron of the fat mass and obesity–associated (FTO) gene, and BMI (P = 4.97 × 10−7) and FFMI (P = 1.19 × 10−7). We replicated the association in a fourth, independent cohort consisting of 502 subjects with COPD from COPDGene (P = 6 × 10−3). Within the largest contributing cohort of our analysis, lung function, as assessed by forced expiratory volume at 1 second, varied significantly by FTO genotype. Our analysis suggests a potential role for the FTO locus in the determination of anthropomorphic measures associated with COPD.
chronic obstructive pulmonary disease genetics; chronic obstructive pulmonary disease epidemiology; chronic obstructive pulmonary disease metabolism; genome-wide association study
Rationale: Chromosome 12p has been linked to chronic obstructive pulmonary disease (COPD) in the Boston Early-Onset COPD Study (BEOCOPD), but a susceptibility gene in that region has not been identified.
Objectives: We used high-density single-nucleotide polymorphism (SNP) mapping to implicate a COPD susceptibility gene and an animal model to determine the potential role of SOX5 in lung development and COPD.
Methods: On chromosome 12p, we genotyped 1,387 SNPs in 386 COPD cases from the National Emphysema Treatment Trial and 424 control smokers from the Normative Aging Study. SNPs with significant associations were then tested in the BEOCOPD study and the International COPD Genetics Network. Based on the human results, we assessed histology and gene expression in the lungs of Sox5−/− mice.
Measurements and Main Results: In the case-control analysis, 27 SNPs were significant at P ≤ 0.01. The most significant SNP in the BEOCOPD replication was rs11046966 (National Emphysema Treatment Trial–Normative Aging Study P = 6.0 × 10−4, BEOCOPD P = 1.5 × 10−5, combined P = 1.7 × 10−7), located 3′ to the gene SOX5. Association with rs11046966 was not replicated in the International COPD Genetics Network. Sox5−/− mice showed abnormal lung development, with a delay in maturation before the saccular stage, as early as E16.5. Lung pathology in Sox5−/− lungs was associated with a decrease in fibronectin expression, an extracellular matrix component critical for branching morphogenesis.
Conclusions: Genetic variation in the transcription factor SOX5 is associated with COPD susceptibility. A mouse model suggests that the effect may be due, in part, to its effects on lung development and/or repair processes.
chronic obstructive pulmonary disease; emphysema; knockout mice; lung development; single nucleotide polymorphism
Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD which address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of loop involvement in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, loop closure necessitates a conformational change of a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria makes the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.
Rationale: Chronic obstructive pulmonary disease (COPD), characterized by airflow limitation, is a disorder with high phenotypic and genetic heterogeneity. Pulmonary emphysema is a major but variable component of COPD; familial data suggest that different components of COPD, such as emphysema, may be influenced by specific genetic factors.
Objectives: To identify genetic determinants of emphysema assessed through high-resolution chest computed tomography in individuals with COPD.
Methods: We performed a genome-wide association study (GWAS) of emphysema determined from chest computed tomography scans with a total of 2,380 individuals with COPD in three independent cohorts of white individuals from (1) a cohort from Bergen, Norway, (2) the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) Study, and (3) the National Emphysema Treatment Trial (NETT). We tested single-nucleotide polymorphism associations with the presence or absence of emphysema determined by radiologist assessment in two of the three cohorts and a quantitative emphysema trait (percentage of lung voxels less than –950 Hounsfield units) in all three cohorts.
Measurements and Main Results: We identified association of a single-nucleotide polymorphism in BICD1 with the presence or absence of emphysema (P = 5.2 × 10−7 with at least mild emphysema vs. control subjects; P = 4.8 × 10−8 with moderate and more severe emphysema vs. control subjects).
Conclusions: Our study suggests that genetic variants in BICD1 are associated with qualitative emphysema in COPD. Variants in BICD1 are associated with length of telomeres, which suggests that a mechanism linked to accelerated aging may be involved in the pathogenesis of emphysema.
Clinical trial registered with www.clinicaltrials.gov (NCT00292552).
emphysema; chronic obstructive pulmonary disease; BICD1; single-nucleotide polymorphism
Rationale: Genome-wide association studies have shown significant associations between variants near hedgehog interacting protein HHIP, FAM13A, and cholinergic nicotinic acetylcholine receptor CHRNA3/5 with increased risk of chronic obstructive pulmonary disease (COPD) in smokers; however, the disease mechanisms behind these associations are not well understood.
Objectives: To identify the association between replicated loci and COPD-related phenotypes in well-characterized patient populations.
Methods: The relationship between these three loci and COPD-related phenotypes was assessed in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-point (ECLIPSE) cohort. The results were validated in the family-based International COPD Genetics Network (ICGN).
Measurements and Main Results: The CHRNA3/5 locus was significantly associated with pack-years of smoking (P = 0.002 and 3 × 10−4), emphysema assessed by a radiologist using high-resolution computed tomography (P = 2 × 10−4 and 4.8 × 10−5), and airflow obstruction (P = 0.004 and 1.8 × 10−5) in the ECLIPSE and ICGN populations, respectively. However, variants in the IREB2 gene were only significantly associated with FEV1. The HHIP locus was not associated with smoking intensity but was associated with FEV1/FVC (P = 1.9 × 10−4 and 0.004 in the ECLIPSE and ICGN populations). The HHIP locus was also associated with fat-free body mass (P = 0.007) and with both retrospectively (P = 0.015) and prospectively (P = 0.024) collected COPD exacerbations in the ECLIPSE cohort. Single-nucleotide polymorphisms in the FAM13A locus were associated with lung function.
Conclusions: The CHRNA3/5 locus was associated with increased smoking intensity and emphysema in individuals with COPD, whereas the HHIP and FAM13A loci were not associated with smoking intensity. The HHIP locus was associated with the systemic components of COPD and with the frequency of COPD exacerbations. FAM13A locus was associated with lung function.
COPD exacerbations; nicotine addiction; high-resolution CT; genetic association analysis; emphysema
Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger regulating diverse cellular functions including motility, biofilm formation, cell cycle progression and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TDB1265 EAL domain was determined in a free state (1.8 Å) and in complex with c-di-GMP (2.35 Å) and unveiled the role of the conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of the c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.
EAL domain; cyclic di-GMP; phosphodiesterase; X-ray crystallography; Thiobacillus denitrificans
Previous expression quantitative trait loci (eQTL) studies have performed genetic association studies for gene expression, but most of these studies examined lymphoblastoid cell lines from non-diseased individuals. We examined the genetics of gene expression in a relevant disease tissue from chronic obstructive pulmonary disease (COPD) patients to identify functional effects of known susceptibility genes and to find novel disease genes. By combining gene expression profiling on induced sputum samples from 131 COPD cases from the ECLIPSE Study with genomewide single nucleotide polymorphism (SNP) data, we found 4315 significant cis-eQTL SNP-probe set associations (3309 unique SNPs). The 3309 SNPs were tested for association with COPD in a genomewide association study (GWAS) dataset, which included 2940 COPD cases and 1380 controls. Adjusting for 3309 tests (p<1.5e-5), the two SNPs which were significantly associated with COPD were located in two separate genes in a known COPD locus on chromosome 15: CHRNA5 and IREB2. Detailed analysis of chromosome 15 demonstrated additional eQTLs for IREB2 mapping to that gene. eQTL SNPs for CHRNA5 mapped to multiple linkage disequilibrium (LD) bins. The eQTLs for IREB2 and CHRNA5 were not in LD. Seventy-four additional eQTL SNPs were associated with COPD at p<0.01. These were genotyped in two COPD populations, finding replicated associations with a SNP in PSORS1C1, in the HLA-C region on chromosome 6. Integrative analysis of GWAS and gene expression data from relevant tissue from diseased subjects has located potential functional variants in two known COPD genes and has identified a novel COPD susceptibility locus.
The EF1143 protein from Enterococcus faecalis is a distant homolog of deoxynucleotide triphosphate triphosphohydrolases (dNTPases) from Escherichia coli and Thermus thermophilus. These dNTPases are important components in the regulation of the dNTP pool in bacteria. Biochemical assays of the EF1143 dNTPase activity demonstrated nonspecific hydrolysis of all canonical dNTPs in the presence of Mn2+. In contrast, with Mg2+ hydrolysis required the presence of dGTP as an effector, activating the degradation of dATP and dCTP with dGTP also being consumed in the reaction with dATP. The crystal structure of EF1143 and dynamic light scattering measurements in solution revealed a tetrameric oligomer as the most probable biologically active unit. The tetramer contains four dGTP specific allosteric regulatory sites and four active sites. Examination of the active site with the dATP substrate suggests an in-line nucleophilic attack on the α-phosphate center as a possible mechanism of the hydrolysis and two highly conserved residues, His-129 and Glu-122, as an acid-base catalytic dyad. Structural differences between EF1143 apo and holo forms revealed mobility of the α3 helix that can regulate the size of the active site binding pocket and could be stabilized in the open conformation upon formation of the tetramer and dGTP effector binding.
Allosteric Regulation; Crystal Structure; Enzyme Catalysis; Enzyme Structure; Metalloenzymes; Nucleoside Nucleotide Metabolism; Phosphodiesterases; Deoxynucleotide Triphosphate Triphosphohydrolase
COPD; Genetics; Association analysis; Consortium
Germanium nanowire (GeNW)-positioned Schottky solar cell was fabricated by a solution process. A GeNW-containing solution was spread out onto asymmetric metal electrodes to produce a rectifying current flow. Under one-sun illumination, the GeNW-positioned Schottky solar cell yields an open-circuit voltage of 177 mV and a short-circuit current of 19.2 nA. Schottky and ohmic contacts between a single GeNW and different metal electrodes were systematically investigated. This solution process may provide a route to the cost-effective nanostructure solar architecture.
ExoU is a potent effector protein that causes rapid host cell death upon injection by the type III secretion system of Pseudomonas aeruginosa. The N-terminal half of ExoU contains a patatin-like phospholipase A2 (PLA2) domain that requires the host cell cofactor superoxide dismutase 1 (SOD1) for activation, while the C-terminal 137 amino acids constitute a membrane localization domain (MLD). Previous studies had utilized insertion and deletion mutations to show that portions of the MLD are required for membrane localization and catalytic activity. Here we further characterize this domain by identifying six residues that are essential for ExoU activity. Substitutions at each of these positions resulted in abrogation of membrane targeting, decreased ExoU-mediated cytotoxicity, and reductions in PLA2 activity. Likewise, each of the six MLD residues was necessary for full virulence in cell culture and murine models of acute pneumonia. Purified recombinant ExoU proteins with substitutions at five of the six residues were not activated by SOD1, suggesting that these five residues are critical for activation by this cofactor. Interestingly, these same five ExoU proteins were partially activated by HeLa cell extracts, suggesting that a host cell cofactor other than SOD1 is capable of modulating the activity of ExoU. These findings add to our understanding of the role of the MLD in ExoU-mediated virulence.
We estimated state-level disability-associated health-care expenditures (DAHE) for the U.S. adult population.
We used a two-part model to estimate DAHE for the noninstitutionalized U.S. civilian adult population using data from the 2002–2003 Medical Expenditure Panel Survey and state-level data from the Behavioral Risk Factor Surveillance System. Administrative data for people in institutions were added to generate estimates for the total adult noninstitutionalized population. Individual-level data on total health-care expenditures along with demographic, socioeconomic, geographic, and payer characteristics were used in the models.
The DAHE for all U.S. adults totaled $397.8 billion in 2006, with state expenditures ranging from $598 million in Wyoming to $40.1 billion in New York. Of the national total, the DAHE were $118.9 billion for the Medicare population, $161.1 billion for Medicaid recipients, and $117.8 billion for the privately insured and uninsured populations. For the total U.S. adult population, 26.7% of health-care expenditures were associated with disability, with proportions by state ranging from 16.9% in Hawaii to 32.8% in New York. This proportion varied greatly by payer, with 38.1% for Medicare expenditures, 68.7% for Medicaid expenditures, and 12.5% for nonpublic health-care expenditures associated with disability.
DAHE vary greatly by state and are borne largely by the public sector, and particularly by Medicaid. Policy makers need to consider initiatives that will help reduce the prevalence of disabilities and disability-related health disparities, as well as improve the lives of people with disabilities.
Substantial evidence suggests that there is genetic susceptibility to chronic obstructive pulmonary disease (COPD). To identify common genetic risk variants, we performed a genome-wide association study in 2940 cases and 1380 smoking controls with normal lung function. We demonstrate a novel susceptibility locus at 4q22.1 in FAM13A (rs7671167, OR=0.76, P=8.6×10−8) and provide evidence of replication in one case-control and two family-based cohorts (for all studies, combined P=1.2×10−11).
Dietary intake of genistein by patients with prostate cancer has been associated with decreased metastasis and mortality. Genistein blocks activation of p38 mitogen-activated protein kinase and thus inhibits matrix metalloproteinase-2 (MMP-2) expression and cell invasion in cultured cells and inhibits metastasis of human prostate cancer cells in mice. We investigated the target for genistein in prostate cancer cells.
Prostate cell lines PC3-M, PC3, 1532NPTX, 1542NPTX, 1532CPTX, and 1542CPTX were used. All cell lines were transiently transfected with a constitutively active mitogen-activated protein kinase kinase 4 (MEK4) expression vector (to increase MEK4 expression), small interfering RNA against MEK4 (to decrease MEK4 expression), or corresponding control constructs. Cell invasion was assessed by a Boyden chamber assay. Gene expression was assessed by a quantitative reverse transcription–polymerase chain reaction. Protein expression was assessed by Western blot analysis. Modeller and AutoDock programs were used for modeling of the structure of MEK4 protein and ligand docking, respectively. MMP-2 transcript levels were assessed in normal prostate epithelial cells from 24 patients with prostate cancer from a phase II randomized trial comparing genistein treatment with no treatment. Statistical significance required a P value of .050 or less. All statistical tests were two-sided.
Overexpression of MEK4 increased MMP-2 expression and cell invasion in all six cell lines. Decreased MEK4 expression had the opposite effects. Modeling showed that genistein bound to the active site of MEK4. Genistein inhibited MEK4 kinase activity with a half maximal inhibitory concentration of 0.40 μM (95% confidence interval [CI] = 0.36 to 0.45 μM). The MMP-2 transcript level in normal prostate epithelial cells was statistically significantly higher in the untreated group (100%) than in the genistein-treated group (24%; difference = 76%, 95% CI = 38% to 115%; P = .045).
We identified MEK4 as a proinvasion protein in six human prostate cancer cell lines and the target for genistein. We showed, to our knowledge for the first time, that genistein treatment, compared with no treatment, was associated with decreased levels of MMP-2 transcripts in normal prostate cells from prostate cancer–containing tissue.
Neurons in superficial (SDH) and deep (DDH) laminae of the spinal cord dorsal horn receive sensory information from skin, muscle, joints and viscera. In both regions, glycine- (GlyR) and GABAA-receptors (GABAARs) contribute to fast synaptic inhibition. For rat, several types of GABAAR coexist in the two regions and each receptor type provides different contributions to inhibitory tone. Recent work in mouse has discovered an additional type of GlyR, (containing alpha 3 subunits) in the SDH. The contribution of differing forms of the GlyR to sensory processing in SDH and DDH is not understood.
Methods and Results
Here we compare fast inhibitory synaptic transmission in mouse (P17-37) SDH and DDH using patch-clamp electrophysiology in transverse spinal cord slices (L3-L5 segments, 23°C). GlyR-mediated mIPSCs were detected in 74% (25/34) and 94% (25/27) of SDH and DDH neurons, respectively. In contrast, GABAAR-mediated mIPSCs were detected in virtually all neurons in both regions (93%, 14/15 and 100%, 18/18). Several Gly- and GABAAR properties also differed in SDH vs. DDH. GlyR-mediated mIPSC amplitude was smaller (37.1 ± 3.9 vs. 64.7 ± 5.0 pA; n = 25 each), decay time was slower (8.5 ± 0.8 vs. 5.5 ± 0.3 ms), and frequency was lower (0.15 ± 0.03 vs. 0.72 ± 0.13 Hz) in SDH vs. DDH neurons. In contrast, GABAAR-mediated mIPSCs had similar amplitudes (25.6 ± 2.4, n = 14 vs. 25. ± 2.0 pA, n = 18) and frequencies (0.21 ± 0.08 vs. 0.18 ± 0.04 Hz) in both regions; however, decay times were slower (23.0 ± 3.2 vs. 18.9 ± 1.8 ms) in SDH neurons. Mean single channel conductance underlying mIPSCs was identical for GlyRs (54.3 ± 1.6 pS, n = 11 vs. 55.7 ± 1.8, n = 8) and GABAARs (22.7 ± 1.7 pS, n = 10 vs. 22.4 ± 2.0 pS, n = 11) in both regions. We also tested whether the synthetic endocanabinoid, methandamide (methAEA), had direct effects on Gly- and GABAARs in each spinal cord region. MethAEA (5 μM) reduced GlyR-mediated mIPSC frequency in SDH and DDH, but did not affect other properties. Similar results were observed for GABAAR mediated mIPSCs, however, rise time was slowed by methAEA in SDH neurons.
Together these data show that Gly- and GABAARs with clearly differing physiological properties and cannabinoid-sensitivity contribute to fast synaptic inhibition in mouse SDH and DDH.
There is considerable variability in the susceptibility of smokers to develop chronic obstructive pulmonary disease (COPD). The only known genetic risk factor is severe deficiency of α1-antitrypsin, which is present in 1–2% of individuals with COPD. We conducted a genome-wide association study (GWAS) in a homogenous case-control cohort from Bergen, Norway (823 COPD cases and 810 smoking controls) and evaluated the top 100 single nucleotide polymorphisms (SNPs) in the family-based International COPD Genetics Network (ICGN; 1891 Caucasian individuals from 606 pedigrees) study. The polymorphisms that showed replication were further evaluated in 389 subjects from the US National Emphysema Treatment Trial (NETT) and 472 controls from the Normative Aging Study (NAS) and then in a fourth cohort of 949 individuals from 127 extended pedigrees from the Boston Early-Onset COPD population. Logistic regression models with adjustments of covariates were used to analyze the case-control populations. Family-based association analyses were conducted for a diagnosis of COPD and lung function in the family populations. Two SNPs at the α-nicotinic acetylcholine receptor (CHRNA 3/5) locus were identified in the genome-wide association study. They showed unambiguous replication in the ICGN family-based analysis and in the NETT case-control analysis with combined p-values of 1.48×10−10, (rs8034191) and 5.74×10−10 (rs1051730). Furthermore, these SNPs were significantly associated with lung function in both the ICGN and Boston Early-Onset COPD populations. The C allele of the rs8034191 SNP was estimated to have a population attributable risk for COPD of 12.2%. The association of hedgehog interacting protein (HHIP) locus on chromosome 4 was also consistently replicated, but did not reach genome-wide significance levels. Genome-wide significant association of the HHIP locus with lung function was identified in the Framingham Heart study (Wilk et al., companion article in this issue of PLoS Genetics; doi:10.1371/journal.pgen.1000429). The CHRNA 3/5 and the HHIP loci make a significant contribution to the risk of COPD. CHRNA3/5 is the same locus that has been implicated in the risk of lung cancer.
There is considerable variability in the susceptibility of smokers to develop chronic obstructive pulmonary disease (COPD), which is a heritable multi-factorial trait. Identifying the genetic determinants of COPD risk will have tremendous public health importance. This study describes the first genome-wide association study (GWAS) in COPD. We conducted a GWAS in a homogenous case-control cohort from Norway and evaluated the top 100 single nucleotide polymorphisms in the family-based International COPD Genetics Network. The polymorphisms that showed replication were further evaluated in subjects from the US National Emphysema Treatment Trial and controls from the Normative Aging Study and then in a fourth cohort of extended pedigrees from the Boston Early-Onset COPD population. Two polymorphisms in the α-nicotinic acetylcholine receptor 3/5 locus on chromosome 15 showed unambiguous evidence of association with COPD. This locus has previously been implicated in both smoking behavior and risk of lung cancer, suggesting the possibility of multiple functional polymorphisms in the region or a single polymorphism with wide phenotypic consequences. The hedgehog interacting protein (HHIP) locus on chromosome 4, which is associated with COPD, is also a significant risk locus for COPD.