Pulmonary function measures are heritable traits that predict morbidity and mortality and define chronic obstructive pulmonary disease (COPD). We tested genome-wide association with forced expiratory volume in 1 s (FEV(1)) and the ratio of FEV(1) to forced vital capacity (FVC) in the SpiroMeta consortium (n = 20,288 individuals of European ancestry). We conducted a meta-analysis of top signals with data from direct genotyping (n </= 32,184 additional individuals) and in silico summary association data from the CHARGE Consortium (n = 21,209) and the Health 2000 survey (n </= 883). We confirmed the reported locus at 4q31 and identified associations with FEV(1) or FEV(1)/FVC and common variants at five additional loci: 2q35 in TNS1 (P = 1.11 x 10(-12)), 4q24 in GSTCD (2.18 x 10(-23)), 5q33 in HTR4 (P = 4.29 x 10(-9)), 6p21 in AGER (P = 3.07 x 10(-15)) and 15q23 in THSD4 (P = 7.24 x 10(-15)). mRNA analyses showed expression of TNS1, GSTCD, AGER, HTR4 and THSD4 in human lung tissue. These associations offer mechanistic insight into pulmonary function regulation and indicate potential targets for interventions to alleviate respiratory disease.

Schizophrenia and bipolar disorder are leading causes of morbidity across all populations, with heritability estimates of approximately 80% indicating a substantial genetic component. Population genetics and genome-wide association studies suggest an overlap of genetic risk factors between these illnesses but it is unclear how this genetic component is divided between common gene polymorphisms, rare genomic copy number variants, and rare gene sequence mutations. We report evidence that the lipid transporter gene ABCA13 is a susceptibility factor for both schizophrenia and bipolar disorder. After the initial discovery of its disruption by a chromosome abnormality in a person with schizophrenia, we resequenced ABCA13 exons in 100 cases with schizophrenia and 100 controls. Multiple rare coding variants were identified including one nonsense and nine missense mutations and compound heterozygosity/homozygosity in six cases. Variants were genotyped in additional schizophrenia, bipolar, depression (n > 1600), and control (n > 950) cohorts and the frequency of all rare variants combined was greater than controls in schizophrenia (OR = 1.93, p = 0.0057) and bipolar disorder (OR = 2.71, p = 0.00007). The population attributable risk of these mutations was 2.2% for schizophrenia and 4.0% for bipolar disorder. In a study of 21 families of mutation carriers, we genotyped affected and unaffected relatives and found significant linkage (LOD = 4.3) of rare variants with a phenotype including schizophrenia, bipolar disorder, and major depression. These data identify a candidate gene, highlight the genetic overlap between schizophrenia, bipolar disorder, and depression, and suggest that rare coding variants may contribute significantly to risk of these disorders.

BACKGROUND: When assessing the efficacy of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy, we routinely evaluate gene transfer in the mouse nose and measure transfection efficiency by assessing transgene-specific mRNA using the real-time (TaqMan) quantitative reverse transcriptase-polymerase chain reaction. TaqMan is traditionally used to quantify expression in whole tissue homogenates, which in the nose would contain many cells types, including respiratory and olfactory epithelium. Only the respiratory epithelium is a satisfactory model for human airway epithelium and therefore CFTR gene transfer should be specifically assessed in respiratory epithelial cells (RECs). METHODS: We have compared laser microdissection, pronase digestion and nasal brushing for:(i) the ability to enrich RECs from the wild-type mouse nose and (ii) the length of time to perform the procedure. Using TaqMan, we subsequently assessed gene transfer in enriched RECs after nasal perfusion of GL67A/pCF1-CFTR complexes in a CF mouse model. RESULTS: Laser microdissection successfully isolated RECs; however, time-consuming sample preparation made this technique unsuitable for high-throughput studies. Pronase digestion was sufficiently rapid but only yielded 19%(range = 13%) RECs (n = 6). The nasal brushing method was superior, yielding 92%(range = 15%) RECs (n = 8) and was equally effective in CF knockout mice (91%, range = 14%, n = 10). Importantly, gene transfer was detectable in brushed RECs from 70% of perfused mice and the number of vector-specific transcripts was comparable to 3.5% of endogenous wild-type Cftr levels. CONCLUSIONS: Isolation of RECs by brushing allows accurate assessment of GTA transfection efficiency in an experimental system that is relevant for CF gene therapy. Copyright (c) 2009 John Wiley & Sons, Ltd.

Objectives: The aim of this work was to establish protein profiles in serum and nasal epithelial cells of Cystic Fibrosis individuals in comparison with controls, asthma and chronic obstructive pulmonary disease patients for specific biomarker signatures identification. Design and Methods: Protein extracts were analysed by Surface Enhanced Laser Desorption/Ionization Time-Of-Flight Mass-Spectrometry (SELDI-TOF-MS). Results: The mass spectra revealed a set of peaks with differential expression in serum and nasal cells among the different groups studied, resulting into peak signatures representative/specific of each pathology. Logistic regressions were applied to those peaks, sensitivity, specificity, Youden's indexes and area under the curve (AUC) of the respective receiver operating characteristic (ROC) curves were compared. Discussion: Multivariate analysis demonstrated that combination of peaks have a better predictive value than the individual ones. These protein signatures may serve as diagnostic/prognostic markers for the studied diseases with common clinical features, or as follow-up assessment markers of therapeutic interventions.

BACKGROUND: Human memory and general cognitive abilities are complex functions of high heritability and wide variability in the population. The brain-derived neurotrophic factor (BDNF) plays an important role in mammalian memory formation. METHODOLOGY / PRINCIPAL FINDING: Based on the identification of genes markedly up-regulated during BDNF-induced synaptic consolidation in the hippocampus, we selected genetic variants that were tested in three independent samples, from Norway and Scotland, of adult individuals examined for cognitive abilities. In all samples, we show that markers in the doublecortin- and calmodulin kinase like 1 (DCLK1) gene, are significantly associated with general cognition (IQ scores) and verbal memory function, resisting multiple testing. DCLK1 is a complex gene with multiple transcripts which vary in expression and function. We show that the short variants are all up-regulated after BDNF treatment in the rat hippocampus, and that they are expressed in the adult human brain (mostly in cortices and hippocampus). We demonstrate that several of the associated variants are located in potential alternative promoter- and cis-regulatory elements of the gene and that they affect BDNF-mediated expression of short DCLK1 transcripts in a reporter system. CONCLUSION: These data present DCLK1 as a functionally pertinent gene involved in human memory and cognitive functions.

BACKGROUND: Induced sputum cytology and protein biomarkers can be employed to assess airways inflammation. Increases in sputum iron have been described in inflammatory lung disease. We hypothesised that other sputum metals may be affected by airway inflammation and investigated their potential value as biomarkers. METHODS: Sputum was obtained from 20 healthy control subjects and patients with inflammatory pulmonary diseases: 23 with cystic fibrosis (CF), 16 with bronchiectasis, 17 with asthma and 23 with COPD, and iron, zinc, manganese and copper were measured. 14 CF patients were also studied through an exacerbation cycle. RESULTS: Sputum zinc and iron were elevated in CF and non- CF bronchiectasis vs. controls (p < 0.001, Zinc; p<0.01 Iron). Manganese was elevated in asthma (p<0.01) and bronchiectasis (p<0.05) versus controls. Copper was elevated in CF vs. controls (p<0.05). Zinc decreased (p<0.01) following treatment of CF exacerbation. In CF subjects zinc levels correlated with other biomarkers. CONCLUSIONS: These results suggest a relationship of high concentrations of total zinc and iron with airway inflammation in CF and non-CF bronchiectasis; longitudinal changes being observed in CF. Further work is required to elucidate potential inflammatory mechanisms related to these observations.

G protein-coupled receptors (GPCRs) form a link between the cell and their environment when signalling pathways are activated upon ligand binding. However, the ligands and functions for many GPCRs remain to be determined. We sought to understand the function of one such orphan, G protein-coupled receptor 50 (GPR50), through identification of protein interactors. GPR50 was previously discovered as a candidate gene for psychiatric illness and lipid metabolism. Here, we identified neurite outgrowth inhibitor NOGO-A as an interacting partner of GPR50 by yeast two-hybrid studies. We confirmed the interaction in mammalian cells and found an enrichment of both Gpr50 and neuronal Nogo-A at the synapse. In contrast to neuronal NOGO-A overexpression, overexpression of GPR50 increased neurite length and filopodia- and lamellipodia-like structures in differentiated Neuroscreen-1 cells. The results are markedly similar to a recent study in Nogo-A KO mice and support the involvement of GPR50 in mental disorders with links to several disease mechanisms.

A clinical programme to assess whether lipid GL67A-mediated gene-transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UKCF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wildtype and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (a) the commonly used short-acting CMV promoter/enhancer or (b) the ubiquitin-C (UbC) promoter. In a study of approximately 400 CF mice, vector-specific CFTR mRNA was detected in nasal epithelial cells of 82% of mice treated with a CMV-plasmid (pCF1-CFTR) and 62%-of mice treated with a UbC-plasmid. We then assessed whether CFTR gene-transfer corrected a panel of CFTR-specific endpoint assays in the murine nose including ion transport, periciliary liquid height and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% towards wild-type values. Within this limitation no significant correction of the CF phenotype was detected. At the currents levels of gene-transfer efficiency achievable with non-viral vectors, the murine nose is of limited value as a stepping stone to human trials.

BACKGROUND: Despite sharing 92% sequence identity, paralogous human translation elongation factor 1 alpha-1 (eEF1A1) and elongation factor 1 alpha-2 (eEF1A2) have different but overlapping functional profiles. This may reflect the differential requirements of the cell-types in which they are expressed and is consistent with complex roles for these proteins that extend beyond delivery of tRNA to the ribosome. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the structural basis of these functional differences, we created and validated comparative three-dimensional (3-D) models of eEF1A1 and eEF1A2 on the basis of the crystal structure of homologous eEF1A from yeast. The spatial location of amino acid residues that vary between the two proteins was thereby pinpointed, and their surface electrostatic and lipophilic properties were compared. None of the variations amongst buried amino acid residues are judged likely to have a major structural effect on the protein fold, or to affect domain-domain interactions. Nearly all the variant surface-exposed amino acid residues lie on one face of the protein, in two proximal but distinct sub-clusters. The result of previously performed mutagenesis in yeast may be interpreted as confirming the importance of one of these clusters in actin-bundling and filament disorganization. Interestingly, some variant residues lie in close proximity to, and in a few cases show differences in interactions with, residues previously inferred to be directly involved in binding GTP/GDP, eEF1Balpha and aminoacyl-tRNA. Additional sequence-based predictions, in conjunction with the 3-D models, reveal likely differences in phosphorylation sites that could reconcile some of the functional differences between the two proteins. CONCLUSIONS: The revelation and putative functional assignment of two distinct sub-clusters on the surface of the protein models should enable rational site-directed mutagenesis, including homologous reverse-substitution experiments, to map surface binding patches onto these proteins. The predicted variant-specific phosphorylation sites also provide a basis for experimental verification by mutagenesis. The models provide a structural framework for interpretation of the resulting functional analysis.