Currently, most analytical methods assume all observed genotypes are correct; however, it is clear that errors may reduce statistical power or bias inference in genetic studies. We propose procedures for estimating error rate in genetic analysis and apply them to study the GeneChip Mapping 10K array, which is a technology that has recently become available and allows researchers to survey over 10,000 SNPs in a single assay. We employed a strategy to estimate the genotype error rate in pedigree data. First, the "dose-response" reference curve between error rate and the observable error number were derived by simulation, conditional on given pedigree structures and genotypes. Second, the error rate was estimated by calibrating the number of observed errors in real data to the reference curve. We evaluated the performance of this method by simulation study and applied it to a data set of 30 pedigrees genotyped using the GeneChip Mapping 10K array. This method performed favorably in all scenarios we surveyed. The dose-response reference curve was monotone and almost linear with a large slope. The method was able to estimate accurately the error rate under various pedigree structures and error models and under heterogeneous error rates. Using this method, we found that the average genotyping error rate of the GeneChip Mapping 10K array was about 0.1%. Our method provides a quick and unbiased solution to address the genotype error rate in pedigree data. It behaves well in a wide range of settings and can be easily applied in other genetic projects. The robust estimation of genotyping error rate allows us to estimate power and sample size and conduct unbiased genetic tests. The GeneChip Mapping 10K array has a low overall error rate, which is consistent with the results obtained from alternative genotyping assays.

OBJECTIVE Recent genome-wide association studies have revealed loci associated with glucose and insulin-related traits. We aimed to characterize 19 such loci using detailed measures of insulin processing, secretion, and sensitivity to help elucidate their role in regulation of glucose control, insulin secretion and/or action. RESEARCH DESIGN AND METHODS We investigated associations of loci identified by the Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC) with circulating proinsulin, measures of insulin secretion and sensitivity from oral glucose tolerance tests (OGTTs), euglycemic clamps, insulin suppression tests, or frequently sampled intravenous glucose tolerance tests in nondiabetic humans (n = 29,084). RESULTS The glucose-raising allele in MADD was associated with abnormal insulin processing (a dramatic effect on higher proinsulin levels, but no association with insulinogenic index) at extremely persuasive levels of statistical significance (P = 2.1 x 10(-71)). Defects in insulin processing and insulin secretion were seen in glucose-raising allele carriers at TCF7L2, SCL30A8, GIPR, and C2CD4B. Abnormalities in early insulin secretion were suggested in glucose-raising allele carriers at MTNR1B, GCK, FADS1, DGKB, and PROX1 (lower insulinogenic index; no association with proinsulin or insulin sensitivity). Two loci previously associated with fasting insulin (GCKR and IGF1) were associated with OGTT-derived insulin sensitivity indices in a consistent direction. CONCLUSIONS Genetic loci identified through their effect on hyperglycemia and/or hyperinsulinemia demonstrate considerable heterogeneity in associations with measures of insulin processing, secretion, and sensitivity. Our findings emphasize the importance of detailed physiological characterization of such loci for improved understanding of pathways associated with alterations in glucose homeostasis and eventually type 2 diabetes.

Genome-wide association studies (GWAS) have been extensively used to study common complex diseases such as coronary artery disease (CAD), revealing 153 suggestive CAD loci, of which at least 46 have been validated as having genome-wide significance. However, these loci collectively explain <10% of the genetic variance in CAD. Thus, we must address the key question of what factors constitute the remaining 90% of CAD heritability. We review possible limitations of GWAS, and contextually consider some candidate CAD loci identified by this method. Looking ahead, we propose systems genetics as a complementary approach to unlocking the CAD heritability and etiology. Systems genetics builds network models of relevant molecular processes by combining genetic and genomic datasets to ultimately identify key "drivers" of disease. By leveraging systems-based genetic approaches, we can help reveal the full genetic basis of common complex disorders, enabling novel diagnostic and therapeutic opportunities.

The unfolded protein response (UPR) is a complex network of sensors and target genes that ensure efficient folding of secretory proteins in the endoplasmic reticulum (ER). UPR activation is mediated by three main sensors, which regulate the expression of hundreds of targets. UPR activation can result in outcomes ranging from enhanced cellular function to cell dysfunction and cell death. How this pathway causes such different outcomes is unknown. Fatty liver disease (steatosis) is associated with markers of UPR activation and robust UPR induction can cause steatosis; however, in other cases, UPR activation can protect against this disease. By assessing the magnitude of activation of UPR sensors and target genes in the liver of zebrafish larvae exposed to three commonly used ER stressors (tunicamycin, thapsigargin and Brefeldin A), we have identified distinct combinations of UPR sensors and targets (i.e. subclasses) activated by each stressor. We found that only the UPR subclass characterized by maximal induction of UPR target genes, which we term a stressed-UPR, induced steatosis. Principal component analysis demonstrated a significant positive association between UPR target gene induction and steatosis. The same principal component analysis showed significant correlation with steatosis in samples from patients with fatty liver disease. We demonstrate that an adaptive UPR induced by a short exposure to thapsigargin prior to challenging with tunicamycin reduced both the induction of a stressed UPR and steatosis incidence. We conclude that a stressed UPR causes steatosis and an adaptive UPR prevents it, demonstrating that this pathway plays dichotomous roles in fatty liver disease.

Glioblastoma is a lethal disease featuring a high proliferation of tumor cells, excessive angiogenesis, and heavy drug resistance. The overall survival of glioblastoma patients has been dismal, even with an intensive standard of care. Recent advances in immune checkpoint blockades are changing the treatment of cancers. However, the efficacy of immune checkpoint blockades in glioblastoma is still unclear. Here we investigated the roles of CD103+ cells in regulating the effect of immune checkpoint blockades in glioblastoma mouse models. Our findings indicated that the murine glioblastoma model was not sensitive to immune checkpoint blockades. Flt3L, a growth factor for CD103+ cells, could significantly increase the number of CD103+ dendritic cells in the murine glioblastoma model and, thus, sensitize murine glioblastoma to immune checkpoint blockades. Downstream analysis indicated that the Flt3L and immune checkpoint blockade combination increased the number of tumor-infiltrating CD8+ cells, decreased immune checkpoint expression, and therefore enhanced the antitumor immune response in the murine glioblastoma model. These findings suggested that Flt3L could enhance the efficacy of immune checkpoint blockades in glioblastoma via expanding CD103+ dendritic cells and downstream antitumor immune response.

Several infrequent genetic polymorphisms in the SERPINA1 gene are known to substantially reduce concentration of alpha1-antitrypsin (AAT) in the blood. Since low AAT serum levels fail to protect pulmonary tissue from enzymatic degradation, these polymorphisms also increase the risk for early onset chronic obstructive pulmonary disease (COPD). The role of more common SERPINA1 single nucleotide polymorphisms (SNPs) in respiratory health remains poorly understood. We present here an agnostic investigation of genetic determinants of circulating AAT levels in a general population sample by performing a genome-wide association study (GWAS) in 1392 individuals of the SAPALDIA cohort. Five common SNPs, defined by showing minor allele frequencies (MAFs) >5%, reached genome-wide significance, all located in the SERPINA gene cluster at 14q32.13. The top-ranking genotyped SNP rs4905179 was associated with an estimated effect of β = -0.068 g/L per minor allele (P = 1.20*10(-12)). But denser SERPINA1 locus genotyping in 5569 participants with subsequent stepwise conditional analysis, as well as exon-sequencing in a subsample (N = 410), suggested that AAT serum level is causally determined at this locus by rare (MAF<1%) and low-frequent (MAF 1-5%) variants only, in particular by the well-documented protein inhibitor S and Z (PI S, PI Z) variants. Replication of the association of rs4905179 with AAT serum levels in the Copenhagen City Heart Study (N = 8273) was successful (P<0.0001), as was the replication of its synthetic nature (the effect disappeared after adjusting for PI S and Z, P = 0.57). Extending the analysis to lung function revealed a more complex situation. Only in individuals with severely compromised pulmonary health (N = 397), associations of common SNPs at this locus with lung function were driven by rarer PI S or Z variants. Overall, our meta-analysis of lung function in ever-smokers does not support a functional role of common SNPs in the SERPINA gene cluster in the general population.

Adiponectin is strongly inversely associated with insulin resistance and type 2 diabetes, but its causal role remains controversial. We used a Mendelian randomization approach to test the hypothesis that adiponectin causally influences insulin resistance and type 2 diabetes. We used genetic variants at the ADIPOQ gene as instruments to calculate a regression slope between adiponectin levels and metabolic traits (up to 31,000 individuals) and a combination of instrumental variables and summary statistics-based genetic risk scores to test the associations with gold-standard measures of insulin sensitivity (2,969 individuals) and type 2 diabetes (15,960 case subjects and 64,731 control subjects). In conventional regression analyses, a 1-SD decrease in adiponectin levels was correlated with a 0.31-SD (95% CI 0.26-0.35) increase in fasting insulin, a 0.34-SD (0.30-0.38) decrease in insulin sensitivity, and a type 2 diabetes odds ratio (OR) of 1.75 (1.47-2.13). The instrumental variable analysis revealed no evidence of a causal association between genetically lower circulating adiponectin and higher fasting insulin (0.02 SD; 95% CI -0.07 to 0.11; N = 29,771), nominal evidence of a causal relationship with lower insulin sensitivity (-0.20 SD; 95% CI -0.38 to -0.02; N = 1,860), and no evidence of a relationship with type 2 diabetes (OR 0.94; 95% CI 0.75-1.19; N = 2,777 case subjects and 13,011 control subjects). Using the ADIPOQ summary statistics genetic risk scores, we found no evidence of an association between adiponectin-lowering alleles and insulin sensitivity (effect per weighted adiponectin-lowering allele: -0.03 SD; 95% CI -0.07 to 0.01; N = 2,969) or type 2 diabetes (OR per weighted adiponectin-lowering allele: 0.99; 95% CI 0.95-1.04; 15,960 case subjects vs. 64,731 control subjects). These results do not provide any consistent evidence that interventions aimed at increasing adiponectin levels will improve insulin sensitivity or risk of type 2 diabetes.

Adeno-associated virus (AAV)-based vectors are promising vehicles for therapeutic gene delivery, including for the treatment for heart failure. It has been demonstrated for each of the AAV serotypes 1 through 8 that inhibition of the proteasome results in increased transduction efficiencies. For AAV9, however, the effect of proteasome inhibitors on in vivo transduction has until now not been evaluated. Here we demonstrate, in a well-established rodent heart failure model, that concurrent treatment with the proteasome inhibitor bortezomib does not enhance the efficacy of AAV9.SERCA2a to improve cardiac function as examined by echocardiography and pressure volume analysis. Western blot analysis of SERCA2a protein and reverse transcription-PCR of SERCA2a mRNA demonstrated that bortezomib had no effect on either endogenous rat SERCA2a levels nor on expression levels of human SERCA2a delivered by AAV9.SERCA2a. Similarly, the number of AAV9 genomes in heart samples was unaffected by bortezomib treatment. Interestingly, whereas transduction of HeLa cells and neonatal rat cardiomyocytes by AAV9 was stimulated by bortezomib, transduction of adult rat cardiomyocytes was inhibited. These results indicate an organ/cell-type-specific effect of proteasome inhibition on AAV9 transduction. A future detailed analysis of the underlying molecular mechanisms promises to facilitate the development of improved AAV vectors.

Understanding the genetic basis of airflow obstruction and smoking behaviour is key to determining the pathophysiology of chronic obstructive pulmonary disease (COPD). We used UK Biobank data to study the genetic causes of smoking behaviour and lung health.

Endothelial to mesenchymal transition (EndMT) plays a major role during development, and also contributes to several adult cardiovascular diseases. Importantly, mesenchymal cells including fibroblasts are prominent in atherosclerosis, with key functions including regulation of: inflammation, matrix and collagen production, and plaque structural integrity. However, little is known about the origins of atherosclerosis-associated fibroblasts. Here we show using endothelial-specific lineage-tracking that EndMT-derived fibroblast-like cells are common in atherosclerotic lesions, with EndMT-derived cells expressing a range of fibroblast-specific markers. In vitro modelling confirms that EndMT is driven by TGF-β signalling, oxidative stress and hypoxia; all hallmarks of atherosclerosis. 'Transitioning' cells are readily detected in human plaques co-expressing endothelial and fibroblast/mesenchymal proteins, indicative of EndMT. The extent of EndMT correlates with an unstable plaque phenotype, which appears driven by altered collagen-MMP production in EndMT-derived cells. We conclude that EndMT contributes to atherosclerotic patho-biology and is associated with complex plaques that may be related to clinical events.

Background: Over 90 regions of the genome have been associated with lung function to date, many of which have also been implicated in chronic obstructive pulmonary disease. Methods: We carried out meta-analyses of exome array data and three lung function measures: forced expiratory volume in one second (FEV 1), forced vital capacity (FVC) and the ratio of FEV 1 to FVC (FEV 1/FVC). These analyses by the SpiroMeta and CHARGE consortia included 60,749 individuals of European ancestry from 23 studies, and 7,721 individuals of African Ancestry from 5 studies in the discovery stage, with follow-up in up to 111,556 independent individuals. Results: We identified significant (P<2·8x10 -7) associations with six SNPs: a nonsynonymous variant in RPAP1, which is predicted to be damaging, three intronic SNPs ( SEC24C, CASC17 and UQCC1) and two intergenic SNPs near to LY86 and FGF10. Expression quantitative trait loci analyses found evidence for regulation of gene expression at three signals and implicated several genes, including TYRO3 and PLAU. Conclusions: Further interrogation of these loci could provide greater understanding of the determinants of lung function and pulmonary disease.

To identify factors that regulate gut microbiota density and the impact of varied microbiota density on health, we assayed this fundamental ecosystem property in fecal samples across mammals, human disease, and therapeutic interventions. Physiologic features of the host (carrying capacity) and the fitness of the gut microbiota shape microbiota density. Therapeutic manipulation of microbiota density in mice altered host metabolic and immune homeostasis. In humans, gut microbiota density was reduced in Crohn's disease, ulcerative colitis, and ileal pouch-anal anastomosis. The gut microbiota in recurrent Clostridium difficile infection had lower density and reduced fitness that were restored by fecal microbiota transplantation. Understanding the interplay between microbiota and disease in terms of microbiota density, host carrying capacity, and microbiota fitness provide new insights into microbiome structure and microbiome targeted therapeutics.

Dietary polyphenols promote memory in models of sleep deprivation (SD), stress, and neurodegeneration. The biological properties of dietary polyphenols greatly depend upon the bioavailability of their phenolic metabolites derivatives, which are modulated by gut microbiota. We recently demonstrated that supplementation with grape-derived bioactive dietary polyphenol preparation (BDPP) improves SD-induced cognitive impairment. This study examined the role of the gut microbiota in the ability of BDPP to prevent memory impairment in response to SD. C57BL6/J mice, treated with antibiotics mix (ABX) or BDPP or both, were sleep-deprived at the end of a fear conditioning training session and fear memory was assessed the next day. Gut microbiota composition was analyzed in fecal samples and BDPP-driven phenolic acid metabolites extraction was measured in plasma. We report that the beneficial effect of BDPP on memory in SD is attenuated by ABX-induced dysbiosis. We identified specific communities of fecal microbiota that are associated with the bioavailability of BDPP-derived phenolic acids, which in turn, are associated with memory promotion. These results suggest the gut microbiota composition significantly affects the bioavailability of phenolic acids that drive the dietary polyphenols' cognitive resilience property. Our findings provide a preclinical model with which to test the causal association of gut microbiota-polyphenols, with the ultimate goal of potential developing dietary polyphenols for the prevention/treatment of cognitive impairment.

Atherosclerotic plaque rupture is the culprit event which underpins most acute vascular syndromes such as acute myocardial infarction. Novel biomarkers of plaque rupture could improve biological understanding and clinical management of patients presenting with possible acute vascular syndromes but such biomarker(s) remain elusive. Investigation of biomarkers in the context of de novo plaque rupture in humans is confounded by the inability to attribute the plaque rupture as the source of biomarker release, as plaque ruptures are typically associated with prompt down-stream events of myocardial necrosis and systemic inflammation.

The enzyme tartrate resistant acid phosphatase (TRAP, two isoforms 5a and 5b) is highly expressed in alveolar macrophages, but its function there is unclear and potent selective inhibitors of TRAP are required to assess functional aspects of the protein. We found higher TRAP activity/expression in lungs of patients with chronic obstructive pulmonary disease (COPD) and asthma compared to controls and more TRAP activity in lungs of mice with experimental COPD or asthma. Stimuli related to asthma and/or COPD were tested for their capacity to induce TRAP. Receptor activator of NF-κb ligand (RANKL) and Xanthine/Xanthine Oxidase induced TRAP mRNA expression in mouse macrophages, but only RANKL also induced TRAP activity in mouse lung slices. Several Au(III) coordination compounds were tested for their ability to inhibit TRAP activity and [Au(4,4'-dimethoxy-2,2'-bipyridine)Cl2][PF6] (AubipyOMe) was found to be the most potent inhibitor of TRAP5a and 5b activity reported to date (IC50 1.3 and 1.8 μM respectively). AubipyOMe also inhibited TRAP activity in murine macrophage and human lung tissue extracts. In a functional assay with physiological TRAP substrate osteopontin, AubipyOMe inhibited mouse macrophage migration over osteopontin-coated membranes. In conclusion, higher TRAP expression/activity are associated with COPD and asthma and TRAP is involved in regulating macrophage migration.

Although the prevalence of chronic obstructive pulmonary disease (COPD) is similar between men and women, current evidence used to support bronchodilator therapy has been generated in therapeutic trials that have predominately enrolled male patients. Here, we determined whether there is any significant sex-related differences in FEV1 responses to ipratropium bromide.

The human cerebral cortex contains many cell types that likely underwent independent functional changes during evolution. However, cell-type-specific regulatory landscapes in the cortex remain largely unexplored. Here we report epigenomic and transcriptomic analyses of the two main cortical neuronal subtypes, glutamatergic projection neurons and GABAergic interneurons, in human, chimpanzee, and rhesus macaque. Using genome-wide profiling of the H3K27ac histone modification, we identify neuron-subtype-specific regulatory elements that previously went undetected in bulk brain tissue samples. Human-specific regulatory changes are uncovered in multiple genes, including those associated with language, autism spectrum disorder, and drug addiction. We observe preferential evolutionary divergence in neuron subtype-specific regulatory elements and show that a substantial fraction of pan-neuronal regulatory elements undergoes subtype-specific evolutionary changes. This study sheds light on the interplay between regulatory evolution and cell-type-dependent gene-expression programs, and provides a resource for further exploration of human brain evolution and function.

Drug resistance is a major challenge for chemotherapy in treating human gastric cancer (GC), as the underlying molecular mechanism of chemoresistance in GC remains unknown. Caveolin-1 (Cav-1) is a scaffold protein of plasma membrane caveolae that acts as a tumor modulator by interacting with several cell signals. In this research, we showed that the survival rate of GC cells to cisplatin (CDDP) increased in the presence of Cav-1. Moreover, Cav-1 overexpression inhibited cisplatin-induced apoptosis and improved the survival rate of GC cells. Cav-1 overexpression and knock-down experiments indicated that Cav-1 expression stimulated wingless-type MMTV integration site (WNTs) pathway through the phosphorylation of LRP6 and dephosphorylation of β-catenin. Cav-1 was positively associated with the increase of WNT downstream target gene Met, which led to the activation of HER2 signaling. Moreover, our results demonstrated that the expression of Cav-1 and Met were positively associated with the resistance of GC cells to cisplatin. Collectively, Cav-1 enhances the cisplatin-resistance of GC cells by activating the WNT signaling pathway and Met-HER2 crosstalk. Understanding the role of Cav-1 in the chemoresistance of GC would help to develop novel therapies for a better treatment outcome of GC patients.

PDGFRα+ mesenchymal progenitor cells are associated with pathological fibro-adipogenic processes. Conversely, a beneficial role for these cells during homeostasis or in response to revascularization and regeneration stimuli is suggested, but remains to be defined. We studied the molecular profile and function of PDGFRα+ cells in order to understand the mechanisms underlying their role in fibrosis versus regeneration. We show that PDGFRα+ cells are essential for tissue revascularization and restructuring through injury-stimulated remodeling of stromal and vascular components, context-dependent clonal expansion, and ultimate removal of pro-fibrotic PDGFRα+-derived cells. Tissue ischemia modulates the PDGFRα+ phenotype toward cells capable of remodeling the extracellular matrix and inducing cell-cell and cell-matrix adhesion, likely favoring tissue repair. Conversely, pathological healing occurs if PDGFRα+-derived cells persist as terminally differentiated mesenchymal cells. These studies support a context-dependent "yin-yang" biology of tissue-resident mesenchymal progenitor cells, which possess an innate ability to limit injury expansion while also promoting fibrosis in an unfavorable environment.

Individuals with cystic fibrosis (CF) often experience gastrointestinal (GI) abnormalities. In recent years, the intestinal microbiome has been postulated as a contributor to the development of CF-associated GI complications, hence representing a potential therapeutic target for treatment. We recently developed a rabbit model of CF, which is shown to manifest many human patient-like pathological changes, including intestinal obstruction. Here, we investigated the feces microbiome in young CF rabbits in the absence of antibiotics treatment. Stool samples were collected from seven- to nine-week-old CF rabbits (n = 7) and age-matched wild-type (WT) rabbits (n = 6). Microbiomes were investigated by iTag sequencing of 16S rRNA genes, and functional profiles were predicted using PICRUSt. Consistent with reports of those in pediatric CF patients, the fecal microbiomes of CF rabbits are of lower richness and diversity than that of WT rabbits, with a marked taxonomic and inferred functional dysbiosis. Our work identified a new CF animal model with the manifestation of intestinal dysbiosis phenotype. This model system may facilitate the research and development of novel treatments for CF-associated gastrointestinal diseases.