Genetic variants in RBFOX3 are associated with sleep latency

Time to fall asleep (sleep latency) is a major determinant of sleep quality. Chronic, long sleep latency is a major characteristic of sleep-onset insomnia and/or delayed sleep phase syndrome. In this study we aimed to discover common polymorphisms that contribute to the genetics of sleep latency. We performed a meta-analysis of genome-wide association studies (GWAS) including 2 572 737 single nucleotide polymorphisms (SNPs) established in seven European cohorts including 4242 individuals. We found a cluster of three highly correlated variants (rs9900428, rs9907432 and rs7211029) in the RNA-binding protein fox-1 homolog 3 gene (RBFOX3) associated with sleep latency (P-values=5.77 × 10-08, 6.59 × 10- 08 and 9.17 × 10- 08). These SNPs were replicated in up to 12 independent populations including 30 377 individuals (P-values=1.5 × 10- 02, 7.0 × 10- 03 and 2.5 × 10- 03; combined meta-analysis P-values=5.5 × 10-07, 5.4 × 10-07 and 1.0 × 10-07). A functional prediction of RBFOX3 based on co-expression with other genes shows that this gene is predominantly expressed in brain (P-value=1.4 × 10-316) and the central nervous system (P-value=7.5 × 10- 321). The predicted function of RBFOX3 based on co-expression analysis with other genes shows that this gene is significantly involved in the release cycle of neurotransmitte

Deciphering the RNA landscape by RNAome sequencing

Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods. © Kasper WJ Derks, Branislav Misovic, Mirjam CGN van den Hout, Christel EM Kockx, Cesar Payan Gomez, Rutger WW Brouwer, Harry Vrieling, Jan HJ Hoeijmakers, Wilfred FJ van IJcken, and Joris Pothof

Deciphering the RNA landscape by RNAome sequencing

Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods. © Kasper WJ Derks, Branislav Misovic, Mirjam CGN van den Hout, Christel EM Kockx, Cesar Payan Gomez, Rutger WW Brouwer, Harry Vrieling, Jan HJ Hoeijmakers, Wilfred FJ van IJcken, and Joris Pothof

Targeted chromatin conformation analysis identifies novel distal neural enhancers of ZEB2 in pluripotent stem cell differentiation

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat-Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2's gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons.status: publishe

Effects of freeze-thawing and intravenous infusion on mesenchymal stromal cell gene expression.

Mesenchymal stromal cells (MSC) are increasingly used as an investigative therapeutic product for immune disorders and degenerative disease. Typically, MSC are isolated from human tissue, expanded in culture and cryopreserved until usage. The safety and efficacy of MSC therapy will depend on the phenotypical and functional characteristics of MSC. The freeze-thawing procedure may change these characteristics. Furthermore, the cells encounter a microenvironment after administration that may impact their properties. It has been demonstrated that the majority of MSC localize to the lungs after intravenous infusion, making this the site to study the effects of the in vivo milieu on administered MSC. In the present study we investigated the effect of freeze-thawing and the mouse lung microenvironment on human adipose tissue-derived MSC. There were effects of freeze-thawing on the whole genome expression profile of MSC, although the effects did not exceed inter-donor differences. There were no major changes in the expression of hemostatic regulators on transcriptional level, but significantly increased expression of procoagulant tissue factor on the surface of thawed adipose MSC, correlating with increased procoagulant activity of thawed cells. Exposure for 2h to the lung microenvironment had a major effect on MSC gene expression and affected several immunological pathways. This indicates that MSC undergo functional changes shortly after infusion and this may influence the efficacy of MSC to modulate inflammatory responses. The results of this study demonstrate that MSC rapidly alter in response to the local milieu and that disease specific conditions may shape MSC after administration

The dystrophin gene and cognitive function in the general population

The aim of our study is to investigate whether single-nucleotide dystrophin gene (DMD) variants associate with variability in cognitive functions in healthy populations. The study included 1240 participants from the Erasmus Rucphen family (ERF) study and 1464 individuals from the Rotterdam Study (RS). The participants whose exomes were sequenced and who were assessed for various cognitive traits were included in the analysis. To determine the association between DMD variants and cognitive ability, linear (mixed) modeling with adjustment for age, sex and education was used. Moreover, Sequence Kernel Association Test (SKAT) was used to test the overall association of the rare genetic variants present in the DMD with cognitive traits. Although no DMD variant surpassed the prespecified significance threshold (PG showed strong association (beta = 1.786, P-value = 2.56 x 10(-4)) with block-design test in the ERF study, while another variant rs1800273: G>A showed suggestive association (beta = -0.465, P-value = 0.002) with Mini-Mental State Examination test in the RS. Both variants are highly conserved, although rs147546024: A>G is an intronic variant, whereas rs1800273: G>A is a missense variant in the DMD which has a predicted damaging effect on the protein. Further gene-based analysis of DMD revealed suggestive association (P-values = 0.087 and 0.074) with general cognitive ability in both cohorts. In conclusion, both single variant and gene-based analyses suggest the existence of variants in the DMD which may affect cognitive functioning in the general populations