Heart failure is a complex clinical syndrome and has become the most common reason for adult hospitalization in developed countries. Two subtypes of heart failure, ischemic heart disease (ISCH) and dilated cardiomyopathy (DCM), have been studied using microarray platforms. However, microarray has limited resolution. Here we applied RNA sequencing (RNA-Seq) to identify gene signatures for heart failure from six individuals, including three controls, one ISCH and two DCM patients. Using genes identified from this small RNA-Seq dataset, we were able to accurately classify heart failure status in a much larger set of 313 individuals. The identified genes significantly overlapped with genes identified via genome-wide association studies for cardiometabolic traits and the promoters of those genes were enriched for binding sites for transcriptions factors. Our results indicate that it is possible to use RNA-Seq to classify disease status for complex diseases such as heart failure using an extremely small training dataset.

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Understanding the influence of gene expression on the molecular mechanisms underpinning human phenotypic diversity is fundamental to being able to predict health outcomes and treat disease. We have carried out whole transcriptome expression analysis on a series of eight normal human postmortem eyes by RNA sequencing. Here we present data showing that ∼80% of the transcriptome is expressed in the posterior layers of the eye and that there is significant differential expression not only between the layers of the posterior part of the eye but also between locations of a tissue layer. These differences in expression also extend to alternative splicing and splicing factors. Differentially expressed genes are enriched for genes associated with psychiatric, immune and cardiovascular disorders. Enrichment categories for gene ontology included ion transport, synaptic transmission and visual and sensory perception. Lastly, allele-specific expression was found to be significant forCFH,C3 andCFB, which are known risk genes for age-related macular degeneration. These expression differences should be useful in determining the underlying biology of associations with common diseases of the human retina, retinal pigment epithelium and choroid and in guiding the analysis of the genomic regions involved in the control of normal gene expression.

Central conducting lymphatic anomaly (CCLA) is one of the complex lymphatic anomalies characterized by dilated lymphatic channels, lymphatic channel dysmotility and distal obstruction affecting lymphatic drainage. We performed whole exome sequencing (WES) of DNA from a four-generation pedigree and examined the consequences of the variant by transfection of mammalian cells and morpholino and rescue studies in zebrafish. WES revealed a heterozygous mutation in EPHB4 (RefSeq NM_004444.4; c.2334 + 1G>C) and RNA-Seq demonstrated that the EPHB4 mutation destroys the normal donor site, which leads to the use of a cryptic splice donor that results in retention of the intervening 12-bp intron sequence. Transient co-expression of the wild-type and mutant EPHB4 proteins showed reduced phosphorylation of tyrosine, consistent with a loss-of-function effect. Zebrafish ephb4a morpholino resulted in vessel misbranching and deformities in the lymphatic vessel development, indicative of possible differentiation defects in lymphatic vessels, mimicking the lymphatic presentations of the patients. Immunoblot analysis using zebrafish lysates demonstrated over-activation of mTORC1 as a consequence of reduced EPHB4 signaling. Strikingly, drugs that inhibit mTOR signaling or RAS-MAPK signaling effectively rescued the misbranching phenotype in a comparable manner. Moreover, knock-in of EPHB4 mutation in HEK293T cells also induced mTORC1 activity. Our data demonstrate the pathogenicity of the identified EPHB4 mutation as a novel cause of CCLA and suggesting that ERK inhibitors may have therapeutic benefits in such patients with complex lymphatic anomalies.

Objective Inappropriate transcriptional activation of innate immunity is a pathologic feature of several cardio-metabolic disorders but little is known of inflammatory modulation of long intergenic non-coding RNAs (lincRNAs) in disease-relevant human tissues. Approach and Results We applied deep RNA sequencing (RNA-seq; >500 million filtered reads per sample) to blood and adipose during low-dose experimental endotoxemia (LPS) in a healthy human, with targeted replication in separate individuals undergoing endotoxemia (n=6), to identify inflammatory lincRNAs. A subset of these lincRNAs was examined for expression in adipocytes and monocytes, modulation in adipose in obesity, and overlap with genome-wide association study signals for inflammatory and cardio-metabolic traits. Of a stringent set of 4,284 lincRNAs, about 11–22% were expressed with 201 and 56 lincRNAs modulated by LPS in blood or adipose, respectively. Tissue-specific expression of a subset of six LPS-lincRNAs was replicated with LPS-modulation confirmed for all three expressed in blood and two of four expressed in adipose. The broader generalizabilty of findings in blood of subject A was confirmed by RNA-seq in seven additional subjects. We confirmed adipocytes and monocytes as potential cell sources of selective LPS-regulated lincRNAs and two of these, linc-DMRT2 (P=0.002) and linc-TP53I13 (P=0.01), were suppressed in adipose in obesity. Finally, we provide examples of LPS-modulated lincRNAs that overlap SNPs that are associated with cardiometabolic traits. Conclusion Our findings provide novel insights into tissue-level, inflammatory transcriptome regulation in cardio-metabolic diseases. These are complementary to more usual approaches limited to interrogation of DNA variations.