Rare variants in neuronal excitability genes influence risk for bipolar disorder.

We sequenced the genomes of 200 individuals from 41 families multiply affected with bipolar disorder (BD) to identify contributions of rare variants to genetic risk. We initially focused on 3,087 candidate genes with known synaptic functions or prior evidence from genome-wide association studies. BD pedigrees had an increased burden of rare variants in genes encoding neuronal ion channels, including subunits of GABAA receptors and voltage-gated calcium channels. Four uncommon coding and regulatory variants also showed significant association, including a missense variant in GABRA6. Targeted sequencing of 26 of these candidate genes in an additional 3,014 cases and 1,717 controls confirmed rare variant associations in ANK3, CACNA1B, CACNA1C, CACNA1D, CACNG2, CAMK2A, and NGF. Variants in promoters and 5' and 3' UTRs contributed more strongly than coding variants to risk for BD, both in pedigrees and in the case-control cohort. The genes and pathways identified in this study regulate diverse aspects of neuronal excitability. We conclude that rare variants in neuronal excitability genes contribute to risk for BD.

Mouse hair cycle expression dynamics modeled as coupled mesenchymal and epithelial oscillators.

The hair cycle is a dynamic process where follicles repeatedly move through phases of growth, retraction, and relative quiescence. This process is an example of temporal and spatial biological complexity. Understanding of the hair cycle and its regulation would shed light on many other complex systems relevant to biological and medical research. Currently, a systematic characterization of gene expression and summarization within the context of a mathematical model is not yet available. Given the cyclic nature of the hair cycle, we felt it was important to consider a subset of genes with periodic expression. To this end, we combined several mathematical approaches with high-throughput, whole mouse skin, mRNA expression data to characterize aspects of the dynamics and the possible cell populations corresponding to potentially periodic patterns. In particular two gene clusters, demonstrating properties of out-of-phase synchronized expression, were identified. A mean field, phase coupled oscillator model was shown to quantitatively recapitulate the synchronization observed in the data. Furthermore, we found only one configuration of positive-negative coupling to be dynamically stable, which provided insight on general features of the regulation. Subsequent bifurcation analysis was able to identify and describe alternate states based on perturbation of system parameters. A 2-population mixture model and cell type enrichment was used to associate the two gene clusters to features of background mesenchymal populations and rapidly expanding follicular epithelial cells. Distinct timing and localization of expression was also shown by RNA and protein imaging for representative genes. Taken together, the evidence suggests that synchronization between expanding epithelial and background mesenchymal cells may be maintained, in part, by inhibitory regulation, and potential mediators of this regulation were identified. Furthermore, the model suggests that impairing this negative regulation will drive a bifurcation which may represent transition into a pathological state such as hair miniaturization.

Mutation Patterns Predict Drug Sensitivity in Acute Myeloid Leukemia.

PURPOSE: The inherent genetic heterogeneity of acute myeloid leukemia (AML) has challenged the development of precise and effective therapies. The objective of this study was to elucidate the genomic basis of drug resistance or sensitivity, identify signatures for drug response prediction, and provide resources to the research community. EXPERIMENTAL DESIGN: We performed targeted sequencing, high-throughput drug screening, and single-cell genomic profiling on leukemia cell samples derived from patients with AML. Statistical approaches and machine learning models were applied to identify signatures for drug response prediction. We also integrated large public datasets to understand the co-occurring mutation patterns and further investigated the mutation profiles in the single cells. The features revealed in the co-occurring or mutual exclusivity pattern were further subjected to machine learning models. RESULTS: We detected genetic signatures associated with sensitivity or resistance to specific agents, and identified five co-occurring mutation groups. The application of single-cell genomic sequencing unveiled the co-occurrence of variants at the individual cell level, highlighting the presence of distinct subclones within patients with AML. Using the mutation pattern for drug response prediction demonstrates high accuracy in predicting sensitivity to some drug classes, such as MEK inhibitors for RAS-mutated leukemia. CONCLUSIONS: Our study highlights the importance of considering the gene mutation patterns for the prediction of drug response in AML. It provides a framework for categorizing patients with AML by mutations that enable drug sensitivity prediction.

Gene expression networks in COPD: microRNA and mRNA regulation.

BACKGROUND: The mechanisms underlying chronic obstructive pulmonary disease (COPD) remain unclear. MicroRNAs (miRNAs or miRs) are small non-coding RNA molecules that modulate the levels of specific genes and proteins. Identifying expression patterns of miRNAs in COPD may enhance our understanding of the mechanisms of disease. A study was undertaken to determine if miRNAs are differentially expressed in the lungs of smokers with and without COPD. miRNA and mRNA expression were compared to enrich for biological networks relevant to the pathogenesis of COPD. METHODS: Lung tissue from smokers with no evidence of obstructive lung disease (n=9) and smokers with COPD (n=26) was examined for miRNA and mRNA expression followed by validation. We then examined both miRNA and mRNA expression to enrich for relevant biological pathways. RESULTS: 70 miRNAs and 2667 mRNAs were differentially expressed between lung tissue from subjects with COPD and smokers without COPD. miRNA and mRNA expression profiles enriched for biological pathways that may be relevant to the pathogenesis of COPD including the transforming growth factor ß, Wnt and focal adhesion pathways. miR-223 and miR-1274a were the most affected miRNAs in subjects with COPD compared with smokers without obstruction. miR-15b was increased in COPD samples compared with smokers without obstruction and localised to both areas of emphysema and fibrosis. miR-15b was differentially expressed within GOLD classes of COPD. Expression of SMAD7, which was validated as a target for miR-15b, was decreased in bronchial epithelial cells in COPD. CONCLUSIONS: miRNA and mRNA are differentially expressed in individuals with COPD compared with smokers without obstruction. Investigating these relationships may further our understanding of the mechanisms of disease.

Integrating the MicroRNome into the study of lung disease.

Over the last 15 years, investigators have identified small noncoding RNAs as regulators of gene expression. One type of noncoding RNAs are termed microRNAs (miRNAs). miRNAs are evolutionary conserved, approximately 22-nucleotide single-stranded RNAs that target genes by inducing mRNA degradation or by inhibiting translation. miRNAs are implicated in many critical cellular processes, including apoptosis, proliferation, and differentiation. Furthermore, it is estimated that miRNAs may be responsible for regulating the expression of nearly one-third of the human genome. Despite the identification of greater than 500 mature miRNAs, very little is known about their biological functions and functional targets. In the last 5 years, researchers have increasingly focused on the functional relevance and role that miRNAs play in the pathogenesis of human disease. miRNAs are known to be important in solid organ and hematological malignancies, heart disease, as potential modulators of the immune response, and organ development. It is anticipated that miRNA analysis will emerge as an important complement to proteomic and genomic studies to further our understanding of disease pathogenesis. Despite the application of genomics and proteomics to the study of human lung disease, few studies have examined miRNA expression. This perspective is not meant to be an exhaustive review of miRNA biology but will provide an overview of both miRNA biogenesis and our current understanding of the role of miRNAs in lung disease as well as a perspective on the importance of integrating this analysis as a tool for identifying and understanding the biological pathways in lung-disease pathogenesis.

A systems approach to understanding human rhinovirus and influenza virus infection.

Human rhinovirus and influenza virus infections of the upper airway lead to colds and the flu and can trigger exacerbations of lower airway diseases including asthma and chronic obstructive pulmonary disease. Novel diagnostic and therapeutic targets are still needed to differentiate between the cold and the flu, since the clinical course of influenza can be severe while that of rhinovirus is usually more mild. In our investigation of influenza and rhinovirus infection of human respiratory epithelial cells, we used a systems approach to identify the temporally changing patterns of host gene expression from these viruses. After infection of human bronchial epithelial cells (BEAS-2B) with rhinovirus, influenza virus or co-infection with both viruses, we studied the time-course of host gene expression changes over three days. We modeled host responses to these viral infections with time and documented the qualitative and quantitative differences in innate immune activation and regulation.

A novel Fanconi anaemia subtype associated with a dominant-negative mutation in RAD51.

Fanconi anaemia (FA) is a hereditary disease featuring hypersensitivity to DNA cross-linker-induced chromosomal instability in association with developmental abnormalities, bone marrow failure and a strong predisposition to cancer. A total of 17 FA disease genes have been reported, all of which act in a recessive mode of inheritance. Here we report on a de novo g.41022153G>A; p.Ala293Thr (NM_002875) missense mutation in one allele of the homologous recombination DNA repair gene RAD51 in an FA-like patient. This heterozygous mutation causes a novel FA subtype, 'FA-R', which appears to be the first subtype of FA caused by a dominant-negative mutation. The patient, who features microcephaly and mental retardation, has reached adulthood without the typical bone marrow failure and paediatric cancers. Together with the recent reports on RAD51-associated congenital mirror movement disorders, our results point to an important role for RAD51-mediated homologous recombination in neurodevelopment, in addition to DNA repair and cancer susceptibility.

Protein and microRNA biomarkers from lavage, urine, and serum in military personnel evaluated for dyspnea.

BACKGROUND: We have identified candidate protein and microRNA (miRNA) biomarkers for dyspnea by studying serum, lavage fluid, and urine from military personnel who reported serious respiratory symptoms after they were deployed to Iraq or Afghanistan. METHODS: Forty-seven soldiers with the complaint of dyspnea who enrolled in the STudy of Active Duty Military Personnel for Environmental Dust Exposure (STAMPEDE) underwent comprehensive pulmonary evaluations at the San Antonio Military Medical Center. The evaluation included fiber-optic bronchoscopy with bronchoalveolar lavage. The clinical findings from the STAMPEDE subjects pointed to seven general underlying diagnoses or findings including airway hyperreactivity, asthma, low diffusivity of carbon monoxide, and abnormal cell counts. The largest category was undiagnosed. As an exploratory study, not a classification study, we profiled proteins or miRNAs in lavage fluid, serum, or urine in this group to look for any underlying molecular patterns that might lead to biomarkers. Proteins in lavage fluid and urine were identified by accurate mass tag (database-driven) proteomics methods while miRNAs were profiled by a hybridization assay applied to serum, urine, and lavage fluid. RESULTS: Over seventy differentially expressed proteins were reliably identified both from lavage and from urine in forty-eight dyspnea subjects compared to fifteen controls with no known lung disorder. Six of these proteins were detected both in urine and lavage. One group of subjects was distinguished from controls by expressing a characteristic group of proteins. A related group of dyspnea subjects expressed a unique group of miRNAs that included one miRNA that was differentially overexpressed in all three fluids studied. The levels of several miRNAs also showed modest but direct associations with several standard clinical measures of lung health such as forced vital capacity or gas exchange efficiency. CONCLUSIONS: Candidate proteins and miRNAs associated with the general diagnosis of dyspnea have been identified in subjects with differing medical diagnoses. Since these markers can be measured in readily obtained clinical samples, further studies are possible that test the value of these findings in more formal classification or case-control studies in much larger cohorts of subjects with specific lung diseases such as asthma, emphysema, or some other well-defined lung disease.

A genetic approach to the prediction of drug side effects: bleomycin induces concordant phenotypes in mice of the collaborative cross.

The antineoplastic drug bleomycin leads to the side effect of pulmonary fibrosis in both humans and mice. We challenged genetically diverse inbred lines of mice from the Collaborative Cross with bleomycin to determine the heritability of this phenotype. Sibling pairs of mice from 40 lines were treated with bleomycin. Lung disease was assessed by scoring lung pathology and by measuring soluble collagen levels in lavage fluid. Serum micro ribonucleic acids (miRNAs) were also measured. Inbred sibling pairs of animals demonstrated high coinheritance of the phenotypes of disease susceptibility or disease resistance. The plasma levels of one miRNA were clearly correlated in sibling mice. The results showed that, as in humans, the lines that comprise the Collaborative Cross exhibited wide genetic variation in response to this drug. This finding suggests that the genetically diverse Collaborative Cross animals may reveal drug effects that might be missed if a study were based on a conventional mouse strain.

Systems biology of interstitial lung diseases: integration of mRNA and microRNA expression changes.

BACKGROUND: The molecular pathways involved in the interstitial lung diseases (ILDs) are poorly understood. Systems biology approaches, with global expression data sets, were used to identify perturbed gene networks, to gain some understanding of the underlying mechanisms, and to develop specific hypotheses relevant to these chronic lung diseases. METHODS: Lung tissue samples from patients with different types of ILD were obtained from the Lung Tissue Research Consortium and total cell RNA was isolated. Global mRNA and microRNA were profiled by hybridization and amplification-based methods. Differentially expressed genes were compiled and used to identify critical signaling pathways and potential biomarkers. Modules of genes were identified that formed a regulatory network, and studies were performed on cultured cells in vitro for comparison with the in vivo results. RESULTS: By profiling mRNA and microRNA (miRNA) expression levels, we found subsets of differentially expressed genes that distinguished patients with ILDs from controls and that correlated with different disease stages and subtypes of ILDs. Network analysis, based on pathway databases, revealed several disease-associated gene modules, involving genes from the TGF-ß, Wnt, focal adhesion, and smooth muscle actin pathways that are implicated in advancing fibrosis, a critical pathological process in ILDs. A more comprehensive approach was also adapted to construct a putative global gene regulatory network based on the perturbation of key regulatory elements, transcription factors and microRNAs. Our data underscores the importance of TGF-ß signaling and the persistence of smooth muscle actin-containing fibroblasts in these diseases. We present evidence that, downstream of TGF-ß signaling, microRNAs of the miR-23a cluster and the transcription factor Zeb1 could have roles in mediating an epithelial to mesenchymal transition (EMT) and the resultant persistence of mesenchymal cells in these diseases. CONCLUSIONS: We present a comprehensive overview of the molecular networks perturbed in ILDs, discuss several potential key molecular regulatory circuits, and identify microRNA species that may play central roles in facilitating the progression of ILDs. These findings advance our understanding of these diseases at the molecular level, provide new molecular signatures in defining the specific characteristics of the diseases, suggest new hypotheses, and reveal new potential targets for therapeutic intervention.

Interleukin-6 contributes to inflammation and remodeling in a model of adenosine mediated lung injury.

BACKGROUND: Chronic lung diseases are the third leading cause of death in the United States due in part to an incomplete understanding of pathways that govern the progressive tissue remodeling that occurs in these disorders. Adenosine is elevated in the lungs of animal models and humans with chronic lung disease where it promotes air-space destruction and fibrosis. Adenosine signaling increases the production of the pro-fibrotic cytokine interleukin-6 (IL-6). Based on these observations, we hypothesized that IL-6 signaling contributes to tissue destruction and remodeling in a model of chronic lung disease where adenosine levels are elevated. METHODOLOGY/PRINCIPAL FINDINGS: We tested this hypothesis by neutralizing or genetically removing IL-6 in adenosine deaminase (ADA)-deficient mice that develop adenosine dependent pulmonary inflammation and remodeling. Results demonstrated that both pharmacologic blockade and genetic removal of IL-6 attenuated pulmonary inflammation, remodeling and fibrosis in this model. The pursuit of mechanisms involved revealed adenosine and IL-6 dependent activation of STAT-3 in airway epithelial cells. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate that adenosine enhances IL-6 signaling pathways to promote aspects of chronic lung disease. This suggests that blocking IL-6 signaling during chronic stages of disease may provide benefit in halting remodeling processes such as fibrosis and air-space destruction.

Benzobicyclooctanes as novel inhibitors of TNF-alpha signaling.

A novel series of TNF-alpha inhibitors based on a benzobicyclooctane scaffold is reported. The compounds display good potency in inhibiting TNF-alpha induced apoptosis and NF kappa B activation. Additionally, they are selective for TNF-alpha as they do not inhibit apoptosis induced by soluble Fas ligand. The compounds described here can act as leads for future medicinal chemistry efforts and may also be useful tools for elucidating the TNF-alpha signaling pathway.