The landscape of GWAS validation; systematic review identifying 309 validated non-coding variants across 130 human diseases.

BACKGROUND: The remarkable growth of genome-wide association studies (GWAS) has created a critical need to experimentally validate the disease-associated variants, 90% of which involve non-coding variants. METHODS: To determine how the field is addressing this urgent need, we performed a comprehensive literature review identifying 36,676 articles. These were reduced to 1454 articles through a set of filters using natural language processing and ontology-based text-mining. This was followed by manual curation and cross-referencing against the GWAS catalog, yielding a final set of 286 articles. RESULTS: We identified 309 experimentally validated non-coding GWAS variants, regulating 252 genes across 130 human disease traits. These variants covered a variety of regulatory mechanisms. Interestingly, 70% (215/309) acted through cis-regulatory elements, with the remaining through promoters (22%, 70/309) or non-coding RNAs (8%, 24/309). Several validation approaches were utilized in these studies, including gene expression (n = 272), transcription factor binding (n = 175), reporter assays (n = 171), in vivo models (n = 104), genome editing (n = 96) and chromatin interaction (n = 33). CONCLUSIONS: This review of the literature is the first to systematically evaluate the status and the landscape of experimentation being used to validate non-coding GWAS-identified variants. Our results clearly underscore the multifaceted approach needed for experimental validation, have practical implications on variant prioritization and considerations of target gene nomination. While the field has a long way to go to validate the thousands of GWAS associations, we show that progress is being made and provide exemplars of validation studies covering a wide variety of mechanisms, target genes, and disease areas.

Systematic single-variant and gene-based association testing of thousands of phenotypes in 394,841 UK Biobank exomes.

Genome-wide association studies have successfully discovered thousands of common variants associated with human diseases and traits, but the landscape of rare variations in human disease has not been explored at scale. Exome-sequencing studies of population biobanks provide an opportunity to systematically evaluate the impact of rare coding variations across a wide range of phenotypes to discover genes and allelic series relevant to human health and disease. Here, we present results from systematic association analyses of 4,529 phenotypes using single-variant and gene tests of 394,841 individuals in the UK Biobank with exome-sequence data. We find that the discovery of genetic associations is tightly linked to frequency and is correlated with metrics of deleteriousness and natural selection. We highlight biological findings elucidated by these data and release the dataset as a public resource alongside the Genebass browser for rapidly exploring rare-variant association results.

SAIGEgds-an efficient statistical tool for large-scale PheWAS with mixed models.

SUMMARY: Phenome-wide association studies (PheWASs) are known to be a powerful tool in discovery and replication of genetic association studies. To reduce the computational burden of PheWAS in the large cohorts, such as the UK Biobank, the SAIGE method has been proposed to control for case-control imbalance and sample relatedness in a tractable manner. However, SAIGE is still computationally intensive when deployed in analyzing the associations of thousands of ICD10-coded phenotypes with whole-genome imputed genotype data. Here, we present a new high-performance statistical R package (SAIGEgds) for large-scale PheWAS using generalized linear mixed models. The package implements the SAIGE method in optimized C++ codes, taking advantage of sparse genotype dosages and integrating the efficient genomic data structure file format. Benchmarks using the UK Biobank White British genotype data (N ≈ 430 K) with coronary heart disease and simulated cases show that the implementation in SAIGEgds is 5-6 times faster than the SAIGE R package. When used in conjunction with high-performance computing clusters, SAIGEgds provides an efficient analysis pipeline for biobank-scale PheWAS. AVAILABILITY AND IMPLEMENTATION: https://bioconductor.org/packages/SAIGEgds; vignettes included. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Are drug targets with genetic support twice as likely to be approved? Revised estimates of the impact of genetic support for drug mechanisms on the probability of drug approval.

Despite strong vetting for disease activity, only 10% of candidate new molecular entities in early stage clinical trials are eventually approved. Analyzing historical pipeline data, Nelson et al. 2015 (Nat. Genet.) concluded pipeline drug targets with human genetic evidence of disease association are twice as likely to lead to approved drugs. Taking advantage of recent clinical development advances and rapid growth in GWAS datasets, we extend the original work using updated data, test whether genetic evidence predicts future successes and introduce statistical models adjusting for target and indication-level properties. Our work confirms drugs with genetically supported targets were more likely to be successful in Phases II and III. When causal genes are clear (Mendelian traits and GWAS associations linked to coding variants), we find the use of human genetic evidence increases approval by greater than two-fold, and, for Mendelian associations, the positive association holds prospectively. Our findings suggest investments into genomics and genetics are likely to be beneficial to companies deploying this strategy.

Mineralocorticoid receptor antagonism reverses diabetes-related coronary vasodilator dysfunction: A unique vascular transcriptomic signature.

Coronary microvascular dysfunction predicts and may be a proximate cause of cardiac dysfunction and mortality in diabetes; however, few effective treatments exist for these conditions. We recently demonstrated that mineralocorticoid receptor (MR) antagonism reversed cardiovascular dysfunction in early-stage obesity/insulin resistance. The mechanisms underlying this benefit of MR antagonism and its relevance in the setting of long-term obesity complications like diabetes; however, remain unclear. Thus, the present study evaluated the impact of MR antagonism on diabetes-related coronary dysfunction and defines the MR-dependent vascular transcriptome in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat recapitulating later stages of human diabetes. OLETF rats were treated with spironolactone (Sp) and compared to untreated OLETF and lean Long-Evans Tokushima Otsuka rats. Sp treatment attenuated diabetes-associated adipose and cardiac inflammation/fibrosis and improved coronary endothelium-dependent vasodilation but did not alter enhanced coronary vasoconstriction, blood pressure, or metabolic parameters in OLETF rats. Further mechanistic studies using RNA deep sequencing of OLETF rat aortas revealed 157 differentially expressed genes following Sp including upregulation of genes involved in the molecular regulation of nitric oxide bioavailability (Hsp90ab1, Ahsa1, Ahsa2) as well as novel changes in α1D adrenergic receptors (Adra1d), cyclooxygenase-2 (Ptgs2), and modulatory factors of these pathways (Ackr3, Acsl4). Further, Ingenuity Pathway Analysis predicted inhibition of upstream inflammatory regulators by Sp and inhibition of 'migration of endothelial cells', 'differentiation of smooth muscle', and 'angiogenesis' biological functions by Sp in diabetes. Thus, this study is the first to define the MR-dependent vascular transcriptome underlying treatment of diabetes-related coronary microvascular dysfunction by Sp.

Inferring a role for methylation of intergenic DNA in the regulation of genes aberrantly expressed in precursor B-cell acute lymphoblastic leukemia.

A complete understanding of the mechanisms involved in the development of pre-B ALL is lacking. In this study, we integrated DNA methylation data and gene expression data to elucidate the impact of aberrant intergenic DNA methylation on gene expression in pre-B ALL. We found a subset of differentially methylated intergenic loci that were associated with altered gene expression in pre-B ALL patients. Notably, 84% of these regions were also bound by transcription factors (TF) known to play roles in differentiation and B-cell development in a lymphoblastoid cell line. Further, an overall downregulation of eRNA transcripts was observed in pre-B ALL patients and these transcripts were associated with the downregulation of putative target genes involved in B-cell migration, proliferation, and apoptosis. The identification of novel putative regulatory regions highlights the significance of intergenic DNA sequences and may contribute to the identification of new therapeutic targets for the treatment of pre-B ALL.

Transcriptomic effects of metformin in skeletal muscle arteries of obese insulin-resistant rats.

We examined the effects of metformin, a commonly used antidiabetic drug, on gene expression in multiple arteries. Specifically, transcriptional profiles of feed arteries and second branch order arterioles in the soleus, gastrocnemius, and diaphragm muscles as well as aortic endothelial scrapes were examined from obese insulin-resistant Otsuka Long-Evans Tokushima Fatty rats treated with ( n = 9) or without ( n = 10) metformin from 20 to 32 weeks of age. Metformin-treated rats exhibited a reduction in body weight, adiposity, and HbA1c ( P < 0.05). The greatest number of differentially expressed genes (FDR < 15%) between those treated with and without metformin was found in the red gastrocnemius 2a arterioles (93 genes), followed by the diaphragm 2a arterioles (62 genes), and soleus 2a arterioles (15 genes). We also found that two genes were differentially expressed in aortic endothelial cells (LETMD1 and HMGCS2, both downregulated), one gene in the gastrocnemius feed artery (BLNK, downregulated), and no genes in the soleus and diaphragm feed arteries and white gastrocnemius 2a arterioles. No single gene was altered by metformin across all vessels examined. This study provides evidence that metformin treatment produces distinct gene expression effects throughout the arterial tree in a rat model of obesity and insulin resistance. Genes whose expression was modulated with metformin do not appear to have a clear connection with its known mechanisms of action. These findings support the notion that vascular gene regulation in response to oral pharmacological therapy, such as metformin, is vessel specific. Impact statement This study provides evidence that metformin treatment produces artery-specific gene expression effects. The genes whose expression was modulated with metformin do not appear to have a clear connection with its known mechanisms of action.

Serum miR-122 may serve as a biomarker for response to direct acting antivirals: effect of paritaprevir/R with dasabuvir or ombitasvir on miR-122 in HCV-infected subjects.

Circulating microRNAs (miRNA) have been intensely investigated as biomarkers in disease and therapy. Several studies have identified miR-122 as an important regulator of HCV replication. The effect of new therapies that directly target the HCV replication life cycle on circulating microRNA levels has not been elucidated. We performed expression profiling of circulating miRNA in serum in subjects treated with HCV direct-acting antiviral agents (DAAs). Serum miRNA levels were evaluated from two studies in HCV GT1-infected treatment-naïve subjects and prior nonresponders to pegylated interferon (pegIFN) and ribavirin (RBV) who received paritaprevir/ritonavir + dasabuvir + RBV for 12 weeks, and in treatment-naïve genotype (GT)1-3-infected subjects who received paritaprevir/ritonavir + ombitasvir ± RBV for 12 weeks. Over 100 different miRNA species were detected in serum. Of these, levels of miR-122 showed the most consistent change in response to treatment across all HCV genotypes. In all subjects, miR-122 showed an average four-fold reduction between baseline and week 2, and remained below baseline through post-treatment week 12 in subjects who achieved sustained virological response. In contrast, in subjects who did not achieve SVR, miR-122 levels began to return to baseline levels after the second week of treatment. The change in miR-122 levels was similar across genotypes, and was comparable with or without RBV. This is the first report comparing expression levels of circulating miRNA in HCV GT1-3 subjects treated with IFN-free combinations of DAAs. The results suggest that serum levels of miR-122 are reduced following treatment in subjects who achieve SVR, and correlate with HCV RNA levels across genotypes.

Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media.

OBJECTIVE: Validate single versus sequential culture media for murine embryo development. DESIGN: Prospective laboratory experiment. SETTING: Assisted Reproduction Laboratory. ANIMALS: Murine embryos. INTERVENTIONS: Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. MAIN OUTCOME MEASURES: On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4',6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. RESULTS: Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = <.0001), hatched, and had significantly more trophoblast cells (P = .005) contributing to the increased total cell number. Also at d5, localization of distinct cytoplasmic UCHL1 and nuclear UCHL3 was found in high-quality hatching blastocysts. Localization of UCHL1 and UCHL3 was diffuse and inappropriately dispersed throughout the cytoplasm in low-quality nonhatching blastocysts. CONCLUSIONS: Single medium yields greater cell numbers, an increased growth rate, and more hatching of murine embryos. Cytoplasmic UCHL1 and nuclear UHCL3 localization patterns were indicative of embryo quality. Our conclusions are limited to murine embryos but one might speculate that single medium may also be more beneficial for human embryo culture. Human embryo studies are needed.

Information Management in Cancer Registries: Evaluating the Needs for Cancer Data Collection and Cancer Research.

Cancer registry data collection involves, at a minimum, collecting data on demographics, tumor characteristics, and treatment. A common, identified, and standardized set of data elements is needed to share data quickly and efficiently with consumers of this data. This project highlights the fact that, there is a need to develop common data elements; Surveys were developed for central cancer registries (CCRs) and cancer researchers (CRs) at NCI-designated Cancer Centers, in order to understand data needs. Survey questions were developed based on the project focus, an evaluation of the research registries and database responses, and systematic review of the literature. Questions covered the following topics: 1) Research, 2) Data collection, 3) Database/ repository, 4) Use of data, 5) Additional data items, 6) Data requests, 7) New data fields, and 8) Cancer registry data set. A review of the surveys indicates that all cancer registries' data are used for public health surveillance, and 96% of the registries indicate the data are also used for research. Data are available online in interactive tables from over 50% of CRs and 87% of CCRs. Some other survey responses indicate that CCR treatment data are not complete for example treatment data, however cancer researchers are interested in treatment variables from CCRs. Cancer registries have many data available for review, but need to examine what data are needed and used by different entities. Cancer Registries can further enhance usage through collaborations and partnerships to connect common interests in the data by making registries visible and accessible.

Integrated methylome and transcriptome analysis reveals novel regulatory elements in pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children under the age of 15. In addition to genetic aberrations, epigenetic modifications such as DNA methylation are altered in cancer and impact gene expression. To identify epigenetic alterations in ALL, genome-wide methylation profiles were generated using the methylated CpG island recovery assay followed by next-generation sequencing. More than 25,000 differentially methylated regions (DMR) were observed in ALL patients with ∼ 90% present within intronic or intergenic regions. To determine the regulatory potential of the DMR, whole-transcriptome analysis was performed and integrated with methylation data. Aberrant promoter methylation was associated with the altered expression of genes involved in transcriptional regulation, apoptosis, and proliferation. Novel enhancer-like sequences were identified within intronic and intergenic DMR. Aberrant methylation in these regions was associated with the altered expression of neighboring genes involved in cell cycle processes, lymphocyte activation and apoptosis. These genes include potential epi-driver genes, such as SYNE1, PTPRS, PAWR, HDAC9, RGCC, MCOLN2, LYN, TRAF3, FLT1, and MELK, which may provide a selective advantage to leukemic cells. In addition, the differential expression of epigenetic modifier genes, pseudogenes, and non-coding RNAs was also observed accentuating the role of erroneous epigenetic gene regulation in ALL.

Exercise-induced differential changes in gene expression among arterioles of skeletal muscles of obese rats.

Using next-generation, transcriptome-wide RNA sequencing (RNA-Seq) technology we assessed the effects of exercise training on transcriptional profiles in skeletal muscle arterioles isolated from the soleus and gastrocnemius muscles of Otsuka Long Evans Tokushima Fatty (OLETF) rats that underwent an endurance exercise training program (EX; n = 13), interval sprint training program (SPRINT; n = 14), or remained sedentary (Sed; n = 12). We hypothesized that the greatest effects of exercise would be in the gastrocnemius arterioles. Results show that EX caused the largest number of changes in gene expression in the soleus and white gastrocnemius 2a arterioles with little to no changes in the feed arteries. In contrast, SPRINT caused substantial changes in gene expression in the feed arteries. IPA canonical pathway analysis revealed 18 pathways with significant changes in gene expression when analyzed across vessels and revealed that EX induces increased expression of the following genes in all arterioles examined: Shc1, desert hedgehog protein (Dhh), adenylate cyclase 4 (Adcy4), G protein binding protein, alpha (Gnat1), and Bcl2l1 and decreased expression of ubiquitin D (Ubd) and cAMP response element modulator (Crem). EX increased expression of endothelin converting enzyme (Ece1), Hsp90b, Fkbp5, and Cdcl4b in four of five arterioles. SPRINT had effects on expression of Crem, Dhh, Bcl2l1, and Ubd that were similar to EX. SPRINT also increased expression of Nfkbia, Hspa5, Tubb 2a and Tubb 2b, and Fkbp5 in all five arterioles and increased expression of Gnat1 in all but the soleus second-order arterioles. Many contractile and/or structural protein genes were increased by SPRINT in the gastrocnemius feed artery, but the same genes exhibited decreased expression in red gastrocnemius arterioles. We conclude that training-induced changes in arteriolar gene expression patterns differ by muscle fiber type composition and along the arteriolar tree.

Exercise training causes differential changes in gene expression in diaphragm arteries and 2A arterioles of obese rats.

We employed next-generation, transcriptome-wide RNA sequencing (RNA-Seq) technology to assess the effects of two different exercise training protocols on transcriptional profiles in diaphragm second-order arterioles (D2a) and in the diaphragm feed artery (DFA) from Otsuka Long Evans Tokushima Fatty (OLETF) rats. Arterioles were isolated from the diaphragm of OLETF rats that underwent an endurance exercise training program (EX; n = 13), interval sprint training program (SPRINT; n = 14), or remained sedentary (Sed; n = 12). Our hypothesis was that exercise training would have similar effects on gene expression in the diaphragm and soleus muscle arterioles because diaphragm blood flow increases during exercise to a similar extent as in soleus. Results reveal that several canonical pathways that were significantly altered by exercise in limb skeletal muscles were not among the pathways significantly changed in the diaphragm arterioles including actin cytoskeleton signaling, role of NFAT in regulation of immune response, protein kinase A signaling, and protein ubiquitination pathway. EX training altered the expression of a smaller number of genes than did SPRINT in the DFA but induced a larger number of genes with altered expression in the D2a than did SPRINT. In fact, FDR differential expression analysis (FDR, 10%) indicated that only two genes exhibited altered expression in D2a of SPRINT rats. Very few of the genes that exhibited altered expression in the DFA or D2a were also altered in limb muscle arterioles. Finally, results indicate that the 2a arterioles of soleus muscle (S2a) from endurance-trained animals and the DFA of SPRINT animals exhibited the largest number of genes with altered expression.

Transcriptomic-Wide Discovery of Direct and Indirect HuR RNA Targets in Activated CD4+ T Cells.

Due to poor correlation between steady state mRNA levels and protein product, purely transcriptomic profiling methods may miss genes posttranscriptionally regulated by RNA binding proteins (RBPs) and microRNAs (miRNAs). RNA immunoprecipitation (RIP) methods developed to identify in vivo targets of RBPs have greatly elucidated those mRNAs which may be regulated via transcript stability and translation. The RBP HuR (ELAVL1) and family members are major stabilizers of mRNA. Many labs have identified HuR mRNA targets; however, many of these analyses have been performed in cell lines and oftentimes are not independent biological replicates. Little is known about how HuR target mRNAs behave in conditional knock-out models. In the present work, we performed HuR RIP-Seq and RNA-Seq to investigate HuR direct and indirect targets using a novel conditional knock-out model of HuR genetic ablation during CD4+ T activation and Th2 differentiation. Using independent biological replicates, we generated a high coverage RIP-Seq data set (>160 million reads) that was analyzed using bioinformatics methods specifically designed to find direct mRNA targets in RIP-Seq data. Simultaneously, another set of independent biological replicates were sequenced by RNA-Seq (>425 million reads) to identify indirect HuR targets. These direct and indirect targets were combined to determine canonical pathways in CD4+ T cell activation and differentiation for which HuR plays an important role. We show that HuR may regulate genes in multiple canonical pathways involved in T cell activation especially the CD28 family signaling pathway. These data provide insights into potential HuR-regulated genes during T cell activation and immune mechanisms.

Placental changes caused by food restriction during early pregnancy in mice are reversible.

In a previous study, 50% calorie restriction in mice from d1.5 to 11.5 of pregnancy resulted in reduced placental weights and areas,relative sparing of labyrinth zone area compared to junctional zone area, and dramatic changes in global gene expression profiles.However, little lasting effect was seen on adult offspring of these pregnancies, with a slight reduction in adiposity in males and some changes in liver gene expression in both sexes. The goals of the present study were to determine whether the placental changes induced by caloric restriction in early pregnancy had permanent, irreversible effects on the placenta, and whether the changes in liver gene expression in adult offspring were present before birth. There were no differences in placental weights or areas, or the areas of individual placental zones near term in mice that had previously been food restricted. Global gene expression profiles at d18.5 were indistinguishable in placentas from control and previously food-restricted mothers. In fetuses from restricted dams at d18.5, liver expression of Gck, a key regulator of glycogen synthesis, was reduced, whereas its expression was increased in livers from adult offspring of restricted dams. Ppara expression was also reduced in fetal livers from restricted dams at d18.5, but not in adult offspring livers. We conclude that alterations in the placenta caused by nutrient restriction in early pregnancy are reversible, and that alterations in gene expression in livers of adult offspring are not a result of changes initiated during pregnancy and maintained through adulthood.

Effects of vendor and genetic background on the composition of the fecal microbiota of inbred mice.

The commensal gut microbiota has been implicated as a determinant in several human diseases and conditions. There is mounting evidence that the gut microbiota of laboratory mice (Mus musculus) similarly modulates the phenotype of mouse models used to study human disease and development. While differing model phenotypes have been reported using mice purchased from different vendors, the composition and uniformity of the fecal microbiota in mice of various genetic backgrounds from different vendors is unclear. Using culture-independent methods and robust statistical analysis, we demonstrate significant differences in the richness and diversity of fecal microbial populations in mice purchased from two large commercial vendors. Moreover, the abundance of many operational taxonomic units, often identified to the species level, as well as several higher taxa, differed in vendor- and strain-dependent manners. Such differences were evident in the fecal microbiota of weanling mice and persisted throughout the study, to twenty-four weeks of age. These data provide the first in-depth analysis of the developmental trajectory of the fecal microbiota in mice from different vendors, and a starting point from which researchers may be able to refine animal models affected by differences in the gut microbiota and thus possibly reduce the number of animals required to perform studies with sufficient statistical power.

Identification of a human papillomavirus-associated oncogenic miRNA panel in human oropharyngeal squamous cell carcinoma validated by bioinformatics analysis of the Cancer Genome Atlas.

High-risk human papillomavirus (HPV) is a causative agent for an increasing subset of oropharyngeal squamous cell carcinomas (OPSCCs), and current evidence supports these tumors as having identifiable risk factors and improved response to therapy. However, the biochemical and molecular alterations underlying the pathobiology of HPV-associated OPSCC (designated HPV(+) OPSCC) remain unclear. Herein, we profile miRNA expression patterns in HPV(+) OPSCC to provide a more detailed understanding of pathologic molecular events and to identify biomarkers that may have applicability for early diagnosis, improved staging, and prognostic stratification. Differentially expressed miRNAs were identified in RNA isolated from an initial clinical cohort of HPV(+/-) OPSCC tumors by quantitative PCR-based miRNA profiling. This oncogenic miRNA panel was validated using miRNA sequencing and clinical data from The Cancer Genome Atlas and miRNA in situ hybridization. The HPV-associated oncogenic miRNA panel has potential utility in diagnosis and disease stratification and in mechanistic elucidation of molecular factors that contribute to OPSCC development, progression, and differential response to therapy.

Genome-wide DNA methylation analysis in precursor B-cells.

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182-200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.

Identification of genes whose expression is altered by obesity throughout the arterial tree.

We used next-generation RNA sequencing (RNA-Seq) technology on the whole transcriptome to identify genes whose expression is consistently affected by obesity across multiple arteries. Specifically, we examined transcriptional profiles of the iliac artery as well as the feed artery, first, second, and third branch order arterioles in the soleus, gastrocnemius, and diaphragm muscles from obese Otsuka Long-Evans Tokushima Fatty (OLETF) and lean Long-Evans Tokushima Otsuka (LETO) rats. Within the gastrocnemius and soleus muscles, the number of genes differentially expressed with obesity tended to increase with increasing branch order arteriole number (i.e., decreasing size of the artery). This trend was opposite in the diaphragm. We found a total of 15 genes that were consistently upregulated with obesity (MIS18A, CTRB1, FAM151B, FOLR2, PXMP4, OAS1B, SREBF2, KLRA17, SLC25A44, SNX10, SLFN3, MEF2BNB, IRF7, RAD23A, LGALS3BP) and five genes that were consistently downregulated with obesity (C2, GOLGA7, RIN3, PCP4, CYP2E1). A small fraction (∼9%) of the genes affected by obesity was modulated across all arteries examined. In conclusion, the present study identifies a select number of genes (i.e., 20 genes) whose expression is consistently altered throughout the arterial network in response to obesity and provides further insight into the heterogeneous vascular effects of obesity. Although there is no known direct function of the majority of 20 genes related to vascular health, the obesity-associated upregulation of SREBF2, LGALS3BP, IRF7, and FOLR2 across all arteries is suggestive of an unfavorable vascular phenotypic alteration with obesity. These data may serve as an important resource for identifying novel therapeutic targets against obesity-related vascular complications.

Novel microRNA correlations in the severely injured.

PURPOSE: Severe injury initiates an inflammatory response that can perpetuate immunological dysfunction, uncontrolled inflammation, and subsequent multisystem organ failure. MicroRNAs (miRNAs) have recently been identified as regulators of this inflammatory response. Our study sought to identify the differential expression of unique miRNAs and their correlations with genes of the Toll-like receptor (TLR) pathways, and clinical parameters in the severely injured. METHODS: Fourteen trauma patients requiring transfusion were prospectively enrolled in this institutional review board-approved study. Inclusion criteria consisted of adult patients deemed clinically to be in hemorrhagic shock necessitating transfusion in the acute phase of their injury care. Peripheral blood samples were obtained after admission to the surgical intensive care unit. Expression of circulating mature miRNA from each patient, as well as from 10 healthy, age-matched controls, was determined and compared using the HiSeq 2500 sequencing system and the R software system. Gene expression of TLR signaling pathways for each patient was examined using custom gene expression polymerase chain reaction arrays. Statistical analyses were performed using general linear models and empirical Bayes methods to determine differential expression and Spearman's nonparametric correlation analysis. RESULTS: Subjects were 21-77 years old (mean, 42), 80% male, Injury Severity Score 11-43 (mean, 26), with 11 blunt and 3 penetrating injuries. Three were intubated and 5 received blood products before arrival. Base deficit upon hospital admission was 3 to 20 (mean, 9). All patients required blood transfusion secondary to blood loss sustained during injury. Survival to discharge was 93%. Controls were 27-64 years old (mean, 40) and 60% male. Sequencing analysis revealed 69 differentially expressed miRNAs (P < .05) in the severely injured. Within the differentially expressed miRNAs, there were 12 direct and 6 indirect correlations with multiple genes involved in the TLR3 and TLR4 signaling pathways. The relationships between these same miRNAs and clinical parameters were also analyzed. We discovered 4 direct correlations with base deficit and HCO3, and 7 indirect correlations involving total fresh frozen plasma transfused, base deficit, HCO3, and PaCO2 levels. CONCLUSION: Differential expression and correlations between miRNAs, genes of the TLR pathways, and clinical parameters are unique findings in the severely injured and may lead to a greater understanding of the regulation of sterile inflammation after severe injury.