End RNA Sequencing Research Articles

RNA-Seq transcriptome profiling of Nile rat livers reveals novel insights on the anti-diabetic mechanisms of Water-Soluble Palm Fruit Extract.

Water-Soluble Palm Fruit Extract (WSPFE) has been shown to confer anti-diabetic effects in the Nile rat (NR) (Arvicanthis niloticus). Liquid and powder WSPFE both deterred diabetes onset in NRs fed a high-carbohydrate (hiCHO) diet, but the liquid form provided better protection. In this study, NRs were fed either a hiCHO diet or the same diet added with liquid or powder WSPFE. Following feeding of the diets for 8weeks, random blood glucose levels were measured to categorize NRs as either diabetes-resistant or diabetes-susceptible, based on a cut-off value of 75mg/dL. Livers were then obtained for Illumina HiSeq 4000 paired end RNA-sequencing (RNA-Seq) and the data were mapped to the reference genome. Consistent with physiological and biochemical parameters, the gene expression data obtained indicated that WSPFE was associated with protection against diabetes. Among hepatic genes upregulated by WSPFE versus controls, were genes related to insulin-like growth factor binding protein, leptin receptor, and processes of hepatic metabolism maintenance, while those downregulated were related to antigen binding, immunoglobulin receptor, inflammation- and cancer-related processes. WSPFE supplementation thus helped inhibit diabetes progression in NRs by increasing insulin sensitivity and reducing both the inflammatory effects of a hiCHO diet and the related DNA-damage compensatory mechanisms contributing to liver disease progression. In addition, the genetic permissiveness of susceptible NRs to develop diabetes was potentially associated with dysregulated compensatory mechanisms involving insulin signaling and oxidative stress over time. Further studies on other NR organs associated with diabetes and its complications are warranted.

Locomotion and brain gene expression exhibit sex-specific non-monotonic dose-response to HFPO-DA during Drosophila melanogaster lifespan

BackgroundLegacy per- and polyfluoroalkyl substances (PFAS), known for their environmental persistence and bio-accumulative properties, have been phased out in the U.S. due to public health concerns. A newer polymerization aid used in the manufacture of some fluoropolymers, hexafluoropropylene oxide-dimer acid (HFPO-DA), has lower reported bioaccumulation and toxicity, but is a potential neurotoxicant implicated in dopaminergic neurodegeneration. ObjectiveWe investigated HFPO-DA’s bio-accumulative potential and sex-specific effects on lifespan, locomotion, and brain gene expression in fruit flies. MethodsWe quantified bioaccumulation of HFPO-DA in fruit flies exposed to 8.7 × 104 µg/L of HFPO-DA in the fly media for 14 days via UHPLC-MS. Long-term effect on lifespan was determined by exposing both sexes to 8.7 × 102 – 8.7 × 105 µg/L of HFPO-DA in media. Locomotion was measured following 3, 7, and 14 days of exposures at 8.7 × 101 – 8.7 × 105 µg/L of HFPO-DA in media, and high-throughput 3′-end RNA-sequencing was used to quantify gene expression in fly brains across the same time points. ResultsBioaccumulation of HFPO-DA in fruit flies was not detected. HFPO-DA-induced effects on lifespan, locomotion, and brain gene expression, and lowest adverse effect level (LOAEL) showed sexually dimorphic patterns. Locomotion scores significantly decreased in at least one dose at all time points for females and only at 3-day exposure for males, while brain gene expression exhibited non-monotonic dose-response. Differentially expressed genes correlated to locomotion scores revealed sex-specific numbers of positively and negatively correlated genes per functional category. ConclusionAlthough HFPO-DA effects on locomotion and survival were significant at doses higher than the US EPA reference dose, the brain transcriptomic profiling reveals sex-specific changes and neurological molecular targets; gene enrichments highlight disproportionately affected categories, including immune response: female-specific co-upregulation suggests potential neuroinflammation. Consistent sex-specific exposure effects necessitate blocking for sex in experimental design during HFPO-DA risk assessment.

Multi-Omic Analysis of 253 Untreated Patients with Waldenström's Macroglobulinemia Reveals Clinically and Genomically Distinct Disease Subtypes and a Model for Disease Progression

Background: Waldenström's Macroglobulinemia (WM) is characterized by recurring mutations in MYD88 (95-97%) and CXCR4 (40%). While these genomic insights have heralded new insights into disease pathogenesis and treatment, a multi-omic approach in a large population of WM patients may further clarify underlying disease biology. Methods: Stranded 150bp paired end RNA sequencing was performed on CD19+ bone marrow (BM) biopsies from 253 treatment-naive WM patients along with paired CD19+CD27- and CD19+CD27+ peripheral blood (PB) cells from 13 healthy donors. For 216 patients, whole exome sequencing of CD19+ BM WM cells with CD19- PB controls was also performed. All p-values were subjected to correction using the false discovery rate. Results: Baseline characteristics were typical of treatment-naive patients. Their median age was 66 years; BM disease involvement 50% ; and serum IgM 3,108 mg/dL. The median follow-up was 7.5 years. MYD88 mutations were identified in 250 (98.8%) patients. Exploratory analysis of MYD88 mutated patients revealed three distinct groups of patients that were observed in a hierarchical clustering analysis shown in Fig 1A. Gene set enrichment analysis revealed distinct B-cell Like (BCL) and Plasma Cell Like (PCL) patient groupings and a third non-BCL/PCL patient group that clustered independently. Non-BCL/PCL patients were more likely to be asymptomatic (36/84; 43%) versus those in the BCL (62/96; 64.6%) and PCL (48/68; 70.6%) groups (p<0.005). Among BCL and PCL grouped patients, important differences in disease presentation were noted including BM involvement (50% vs. 70%; p<0.001), adenopathy (18.6% vs. 48.4%; p<0.002), CD10 (1.1% vs. 13.4%; p=0.012), CD23 (39.4% vs. 22.4%; p=0.09) and CD5 (19.1% vs. 6%; p=0.09) expression, respectively. CXCR4 mutations were identified in 76/97 (78.4%) of BCL versus 6/69 (8.7%) of PCL patients (p<0.001). Amplifications in chr18q amplifications were also more prevalent in BCL (14/83; 16.9%) versus PCL (1/64; 1.6%) grouped patients (p=0.035). chr6q deletions were observed in 24/83 (29%) of BCL versus 30/64 (47%) PCL patients (p=0.11). BCL patients also exhibited fewer chr6p amplifications, i.e. 3/83 (3.6%) versus 12/64 (18.8%) of PCL patients (p=0.036). Differences in deletions in chr17p (0/83; 0%) vs (6/64; 9.4% (p=0.036); as well as NOTCH1 (0/82; 0%) vs. (6/64; 9.4%) (p=0.084) and EP300 3/82; 3.7%) vs. (11/64; 17.2%) (p=0.084) mutations were also identified in BCL vs PCL patients, respectively. NOTCH pathway mutations were also found in fewer BCL (17/82; 20.7%) versus PCL (30/64; 46.9%) patients (p=0.01). Diffusion mapping and pseudo-time analysis were performed on the top 1,000 high variance genes which bifurcated patients into two distinct populations over time as shown in Fig. 1B, with the point size scaled for BM involvement. By diffusion pseudo-time (DPT), patients were stratified into early- and late-stage WM, the former showing lower median BM involvement (30% vs. 70%; p<0.001) and serum IgM (2,666 vs. 3713; p=0.133); higher hemoglobin (12.1g/dL vs. 10.65g/dL; p<0.001), serum IgA (56.5mg/dL vs. 41mg/dL; p=0.018) and IgG (629mg/dL vs. 498mg/dL; p=0.015), and less splenomegaly (3.7% vs. 17.2%; p=0.006). A longer time to first treatment (5.3 vs 0.9 months; (p<0.001) was also observed in those with early- versus late- stage WM, respectively. DPT showed that the non-BCL/PCL patients were more likely to be early stage (68/84; 81%) versus BCL (51/97; 53%) and PCL (30/69; 43%) grouped patients (p<0.001). In addition, hierarchical clustering delineated the non-BCL/PCL group into two subgroups with CXCR4 mutations rates of 30/57 (52.6%) and 2/27 (7.4%), and gene expression similarities that aligned with either the BCL or PCL subtypes, respectively. Taken together, these findings suggest that the non-BCL/PCL group represents an earlier evolving stage of WM that may progress into BCL and PCL like WM disease. Conclusions: In this largest multi-omic investigation undertaken into WM, we identified three distinct WM subtypes with distinct clinical and genomic features. Central these findings were the identification of BCL, PCL, and an earlier non-BCL/PCL evolving stage recognized through DPT modeling. The discovery of the DPT model for WM disease progression provides insight into the biology and targeting of WM progression. Our findings provide a critical framework for multi-omic based study and targeted treatment approach to WM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prenatal exposure to particulate matter and placental gene expression.

Background:While strong evidence supports adverse maternal and offspring consequences of air pollution, mechanisms that involve the placenta, a key part of the intrauterine environment, are largely unknown. Previous studies of air pollution and placental gene expression were small candidate gene studies that rarely considered prenatal windows of exposure or the potential role of offspring sex. We examined overall and sex-specific associations of prenatal exposure to fine particulate matter (PM2.5) with genome-wide placental gene expression.Methods:Participants with placenta samples, collected at birth, and childhood health outcomes from CANDLE (Memphis, TN) (n = 776) and GAPPS (Seattle, WA) (n = 205) cohorts of the ECHO-PATHWAYS Consortium were included in this study. PM2.5 exposures during trimesters 1, 2, 3, and the first and last months of pregnancy, were estimated using a spatiotemporal model. Cohort-specific linear adjusted models were fit for each exposure window and expression of >11,000 protein coding genes from paired end RNA sequencing data. Models with interaction terms were used to examine PM2.5-offspring sex interactions. False discovery rate (FDR < 0.10) was used to correct for multiple testing.Results:Mean PM2.5 estimate was 10.5–10.7 μg/m3 for CANDLE and 6.0–6.3 μg/m3 for GAPPS participants. In CANDLE, expression of 13 (11 upregulated and 2 downregulated), 20 (11 upregulated and 9 downregulated) and 3 (2 upregulated and 1 downregulated) genes was associated with PM2.5 in the first trimester, second trimester, and first month, respectively. While we did not find any statistically significant association, overall, between PM2.5 and gene expression in GAPPS, we found offspring sex and first month PM2.5 interaction for DDHD1 expression (positive association among males and inverse association among females). We did not observe PM2.5 and offspring sex interactions in CANDLE.Conclusion:In CANDLE, but not GAPPS, we found that prenatal PM2.5 exposure during the first half of pregnancy is associated with placental gene expression.

A 3'-end capture sequencing method for high-throughput targeted gene expression profiling.

Molecular phenotyping through shallow 3'-end RNA-sequencing workflows is increasingly applied in the context of large-scale chemical or genetic perturbation screens to study disease biology or support drug discovery. While these workflows enable accurate quantification of the most abundant genes, they are less effective for applications that require expression profiling of low abundant transcripts, like long noncoding RNAs (lncRNAs), or selected gene panels. To tackle these issues, we describe a workflow combining 3'-end library preparation with 3'-end hybrid capture probes and shallow RNA-sequencing for cost-effective, targeted quantification of subsets of (low abundant) genes across hundreds to thousands of samples. To assess the performance of the method, we designed a capture probe set for more than 100 mRNA and lncRNA target genes and applied the workflow to a cohort of 360 samples. When compared to standard 3'-end RNA-sequencing, 3'-end capture sequencing resulted in a more than 200-fold enrichment of target gene abundance while conserving relative intergene and intersample abundances. 3'-end RNA capture sequencing enables accurate targeted gene expression profiling at extremely shallow sequencing depth.

Transcriptome in paraffin samples for the diagnosis and prognosis of adrenocortical carcinoma.

DesignMolecular classification is important for the diagnosis and prognosis of adrenocortical tumors (ACT). Transcriptome profiles separate adrenocortical adenomas ‘C2’ from carcinomas, and identify two groups of carcinomas ‘C1A’ and ‘C1B’, of poor and better prognosis respectively. However, many ACT cannot be profiled because of improper or absent freezing procedures, a mandatory requirement so far. The main aim was to determine transcriptome profiles on formalin-fixed paraffin-embedded (FFPE) samples, using the new 3’-end RNA-sequencing technology. A secondary aim was to demonstrate the ability of this technique to explore large FFPE archives, by focusing on the rare oncocytic ACT variants.MethodsWe included 131 ACT: a training cohort from Cochin hospital and an independent validation cohort from Wuerzburg hospital. The 3’ transcriptome was generated from FFPE samples using QuantSeq (Lexogen, Vienna, Austria) and NextSeq500 (Illumina, San Diego, CA, USA).ResultsIn the training cohort, unsupervised clustering identified three groups: ‘C1A’ aggressive carcinomas (n = 28, 29%), ‘C1B’ more indolent carcinomas (n = 28, 29%), and ‘C2’ adenomas (n = 39, 41%). The prognostic value of FFPE transcriptome was confirmed in the validation cohort (5-year OS: 26% in ‘C1A’ (n = 26) and 100% in ‘C1B’ (n = 10), P = 0.003). FFPE transcriptome was an independent prognostic factor in a multivariable model including tumor stage and Ki-67 (OS HR: 7.5, P = 0.01). Oncocytic ACT (n = 19) did not form any specific cluster. Oncocytic carcinomas (n = 6) and oncocytic ACT of uncertain malignant potential (n = 4) were all in ‘C1B’.ConclusionsThe 3’ RNA-sequencing represents a convenient solution for determining ACT molecular class from FFPE samples. This technique should facilitate routine use and large retrospective studies.

Discovery of clinically relevant fusions in pediatric cancer

BackgroundPediatric cancers typically have a distinct genomic landscape when compared to adult cancers and frequently carry somatic gene fusion events that alter gene expression and drive tumorigenesis. Sensitive and specific detection of gene fusions through the analysis of next-generation-based RNA sequencing (RNA-Seq) data is computationally challenging and may be confounded by low tumor cellularity or underlying genomic complexity. Furthermore, numerous computational tools are available to identify fusions from supporting RNA-Seq reads, yet each algorithm demonstrates unique variability in sensitivity and precision, and no clearly superior approach currently exists. To overcome these challenges, we have developed an ensemble fusion calling approach to increase the accuracy of identifying fusions.ResultsOur Ensemble Fusion (EnFusion) approach utilizes seven fusion calling algorithms: Arriba, CICERO, FusionMap, FusionCatcher, JAFFA, MapSplice, and STAR-Fusion, which are packaged as a fully automated pipeline using Docker and Amazon Web Services (AWS) serverless technology. This method uses paired end RNA-Seq sequence reads as input, and the output from each algorithm is examined to identify fusions detected by a consensus of at least three algorithms. These consensus fusion results are filtered by comparison to an internal database to remove likely artifactual fusions occurring at high frequencies in our internal cohort, while a “known fusion list” prevents failure to report known pathogenic events. We have employed the EnFusion pipeline on RNA-Seq data from 229 patients with pediatric cancer or blood disorders studied under an IRB-approved protocol. The samples consist of 138 central nervous system tumors, 73 solid tumors, and 18 hematologic malignancies or disorders. The combination of an ensemble fusion-calling pipeline and a knowledge-based filtering strategy identified 67 clinically relevant fusions among our cohort (diagnostic yield of 29.3%), including RBPMS-MET, BCAN-NTRK1, and TRIM22-BRAF fusions. Following clinical confirmation and reporting in the patient’s medical record, both known and novel fusions provided medically meaningful information.ConclusionsThe EnFusion pipeline offers a streamlined approach to discover fusions in cancer, at higher levels of sensitivity and accuracy than single algorithm methods. Furthermore, this method accurately identifies driver fusions in pediatric cancer, providing clinical impact by contributing evidence to diagnosis and, when appropriate, indicating targeted therapies.

C-Terminal BRE Overexpression in 11q23-Rearranged and t(8;16) Acute Myeloid Leukemia Is Caused By Intragenic Transcription Initiation

Abstract TextOverexpression of the BRE (Brain and Reproductive organ-Expressed ) gene defines a distinct pediatric and adult acute myeloid leukemia (AML) subgroup (Balgobind et al, Leukemia 2010, and Noordermeer et al, Blood 2011 and 2012). BRE overexpression specifically associates with 11q23/MLL/KMT2A-rearranged AML, occurring in 70% and 50% of 11q23 childhood and adult AML cases, respectively. In adult 11q23-rearranged AML, BRE overexpression significantly associates with younger age. BRE functions in BRCA1-A (Breast Cancer 1) complex mediated DNA damage repair, BRISC (BRCC3 isopeptidase complex) complex mediated mitotic spindle assembly and in protection from apoptosis and senescence. Another constituent of the BRCA1-A/BRISC complexes is BRCC3 which is recurrently mutated in AML, especially in cases with a TET2 mutation (Huang et al., Haematologica, 2015). BRE is relatively highly expressed in blood cells in general but its exact function in hematopoiesis is unknown.By performing genome-wide ChIP-sequencing analyses on primary KMT2A-MLLT3 patient material (n=9) we identified a promoter enriched for active chromatin marks (H3K4me3/H3K27Ac) in BRE intron 4, exclusively in AML cases with BRE overexpression. Both 5' RACE end RNA-sequencing identified strong biallelic expression of a previously unknown C-terminal BRE transcript. This transcript starts near the intragenic promoter, with BRE intron 4 sequences spliced to exon 5 and downstream sequences. RNA-seq and qRT-PCR showed that the downstream transcript is expressed 5-8-fold higher compared to the full-length transcript, explaining BRE overexpression. Of note, two start codons in exon 5 of the downstream BRE transcript may cause the formation of N-terminally truncated BRE isoforms.By performing a qRT-PCR that specifically recognizes the new BRE transcript on samples from a large cohort of pediatric and adult AML (n=195) we observed that it was highly expressed in 60% of 11q23/ KMT2A -rearranged (n=58) and 70% of t(8;16)/ KAT6A-CREBBP cases (n=20), while it was virtually absent from other AML subsets (n=101) and normal hematopoietic tissues (n=14). In gene reporter assays, the AML-specific fusion protein KMT2A-MLLT3 transactivated the intragenic BRE promoter. Further epigenome analyses revealed 97 additional intragenic promoter marks frequently bound by KMT2A in AML with C-terminal BRE overexpression. The bound genes may be part of a context-dependent KMT2A-MLLT3-driven oncogenic program, because they were higher expressed in this AML subtype compared to other cytogenetically-defined AML groups (p<0.01). C-terminal BRE might be a contributor to this oncogenic program because in a case with relapsed AML, we observed an ins(11;2) fusing CHORD1 in frame to BRE exon 5. The CHORDC1 fusion to BRE exon 5 is remarkable because the alternate BRE transcript starts with exon 1b also fused to exon 5. This means that if the CHORDC1-BRE and alternate BRE transcripts would be translated, they would contain a similar C-terminal part of the BRE protein. The CHORDC1 disruption may also contribute to AML pathogenesis because it was recently shown that heterozygous Chord1 (a.k.a. Morgana) inactivation in mice results in the spontaneous development of atypical chronic myeloid leukemia (Di Savino, Blood, 2015). We conclude that abnormal expression of C-terminal alternate BRE sequences specific for AML is caused by distinct, non-random events; by a chromosomal abnormality and by intragenic transcription initiation in 11q23/ KMT2A -rearranged and t(8;16)/ KAT6A-CREBBP AML. These findings warrant further studies to determine whether leukemia-specific alternate BRE contributes to disease pathogenesis and how abnormal intragenic transcription activation in AML is brought about. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

MP3RNA-seq: Massively parallel 3' end RNA sequencing for high-throughput gene expression profiling and genotyping.

Transcriptome deep sequencing (RNA-seq) has become a routine method for global gene expression profiling. However, its application to large-scale experiments remains limited by cost and labor constraints. Here we describe a massively parallel 3' end RNA-seq (MP3RNA-seq) method that introduces unique sample barcodes during reverse transcription to permit sample pooling immediately following this initial step. MP3RNA-seq allows for handling of hundreds of samples in a single experiment, at a cost of about $6 per sample for library construction and sequencing. MP3RNA-seq is effective for not only high-throughput gene expression profiling, but also genotyping. To demonstrate its utility, we applied MP3RNA-seq to 477 double haploid lines of maize. We identified 19,429 genes expressed in at least 50% of the lines and 35,836 high-quality single nucleotide polymorphisms for genotyping analysis. Armed with these data, we performed expression and agronomic trait quantitative trait locus (QTL) mapping and identified 25,797 expression QTLs for 15,335 genes and 21 QTLs for plant height, ear height, and relative ear height. We conclude that MP3RNA-seq is highly reproducible, accurate, and sensitive for high-throughput gene expression profiling and genotyping, and should be generally applicable to most eukaryotic species.

High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii.

Toxoplasma gondii is a common parasite of humans and animals, causing life-threatening disease in the immunocompromized, fetal abnormalities when contracted during gestation, and recurrent ocular lesions in some patients. Central to the prevalence and pathogenicity of this protozoan is its ability to adapt to a broad range of environments, and to differentiate between acute and chronic stages. These processes are underpinned by a major rewiring of gene expression, yet the mechanisms that regulate transcription in this parasite are only partially characterized. Deciphering these mechanisms requires a precise and comprehensive map of transcription start sites (TSSs); however, Toxoplasma TSSs have remained incompletely defined. To address this challenge, we used 5′-end RNA sequencing to genomically assess transcription initiation in both acute and chronic stages of Toxoplasma. Here, we report an in-depth analysis of transcription initiation at promoters, and provide empirically-defined TSSs for 7603 (91%) protein-coding genes, of which only 1840 concur with existing gene models. Comparing data from acute and chronic stages, we identified instances of stage-specific alternative TSSs that putatively generate mRNA isoforms with distinct 5′ termini. Analysis of the nucleotide content and nucleosome occupancy around TSSs allowed us to examine the determinants of TSS choice, and outline features of Toxoplasma promoter architecture. We also found pervasive divergent transcription at Toxoplasma promoters, clustered within the nucleosomes of highly-symmetrical phased arrays, underscoring chromatin contributions to transcription initiation. Corroborating previous observations, we asserted that Toxoplasma 5′ leaders are among the longest of any eukaryote studied thus far, displaying a median length of approximately 800 nucleotides. Further highlighting the utility of a precise TSS map, we pinpointed motifs associated with transcription initiation, including the binding sites of the master regulator of chronic-stage differentiation, BFD1, and a novel motif with a similar positional arrangement present at 44% of Toxoplasma promoters. This work provides a critical resource for functional genomics in Toxoplasma, and lays down a foundation to study the interactions between genomic sequences and the regulatory factors that control transcription in this parasite.

Distinct whole-blood transcriptome profile of children with metabolic healthy overweight/obesity compared to metabolic unhealthy overweight/obesity.

Youth populations with overweight/obesity (OW/OB) exhibit heterogeneity in cardiometabolic health phenotypes. The underlying mechanisms for those differences are still unclear. This study aimed to analyze the whole-blood transcriptome profile (RNA-seq) of children with metabolic healthy overweight/obesity (MHO) and metabolic unhealthy overweight/obesity (MUO) phenotypes. Twenty-seven children with OW/OB (10.1 ± 1.3 years, 59% boys) from the ActiveBrains project were included. MHO was defined as having none of the following criteria for metabolic syndrome: elevated fasting glucose, high serum triglycerides, low high-density lipoprotein-cholesterol, and high systolic or diastolic blood pressure, while MUO was defined as presenting one or more of these criteria. Inflammatory markers were additionally determined. Total blood RNA was analyzed by 5'-end RNA-sequencing. Whole-blood transcriptome analysis revealed a distinct pattern of gene expression in children with MHO compared to MUO children. Thirty-two genes differentially expressed were linked to metabolism, mitochondrial, and immune functions. The identified gene expression patterns related to metabolism, mitochondrial, and immune functions contribute to a better understanding of why a subset of the population remains metabolically healthy despite having overweight/obesity. A distinct pattern of whole-blood transcriptome profile (RNA-seq) was identified in children with metabolic healthy overweight/obesity (MHO) compared to metabolic unhealthy overweight/obesity (MUO) phenotype. The most relevant genes in understanding the molecular basis underlying the MHO/MUO phenotypes in children could be: RREB1, FAM83E, SLC44A1, NRG1, TMC5, CYP3A5, TRIM11, and ADAMTSL2. The identified whole-blood transcriptome profile related to metabolism, mitochondrial, and immune functions contribute to a better understanding of why a subset of the population remains metabolically healthy despite having overweight/obesity.

Prenatal Exposure to Particulate Matter and Placental Gene Expression

Background/Aim: Previous studies of air pollution and placental gene expression were small, candidate gene studies. Further, few considered prenatal windows of exposure and the role of offspring sex. We examined overall and sex-specific associations of prenatal maternal exposure to fine particulate matter (PM2.5) with genome-wide placental gene expression. Methods: Participants with placenta samples and childhood health outcomes from the CANDLE (Memphis, TN) (n=428) and GAPPS (Seattle, WA) (n=114) cohorts of the ECHO PATHWAYS Consortium were included. Exposures to PM2.5 during trimesters 1, 2, 3, and, the first and last months of pregnancy were estimated using a spatiotemporal model. Cohort-specific linear models were fit for each exposure window and individual gene expression (&gt;11,000 coding genes) from paired end RNA sequencing data, adjusted for batch, age, race, offspring sex, season, calendar year of birth, gestational age, mode of delivery, presence/absence of labor, smoking, education, and pre-pregnancy obesity. Models with interaction terms were used to examine sex-specific associations. A false discovery rate (FDR&lt;0.10) was used to correct for multiple testing. Results: Average maternal age and gestational age at delivery were 31-32 years and 38 weeks, respectively. In CANDLE, expression of 41 genes (including SCUBE2, PAEP, PGR, and DKK1) was associated with PM2.5 in the first month of pregnancy while expression of YIPF5 was associated with PM2.5 in the final month of pregnancy. In GAPPS, expression of NBPF9 and IGFBP1 was associated with PM2.5 in the final month of the pregnancy. Offspring sex and third trimester PM2.5 exposure interactions were observed for 29 genes (including ETNK2, CRIM1, and VPS35) in CANDLE and two genes (MARCO and HAPLN3) in GAPPS. Conclusions: We found evidence suggesting association between prenatal PM2.5 exposure, particularly during early and late pregnancy, and placental gene expression (including genes involved in signaling and hormone metabolism). Child sex may modify the association.

Genome-wide analysis of the H3K27me3 epigenome and transcriptome in Brassica rapa.

BackgroundGenome-wide maps of histone modifications have been obtained for several plant species. However, most studies focus on model systems and do not enforce FAIR data management principles. Here we study the H3K27me3 epigenome and associated transcriptome of Brassica rapa, an important vegetable cultivated worldwide.FindingsWe performed H3K27me3 chromatin immunoprecipitation followed by high-throughput sequencing and transcriptomic analysis by 3′-end RNA sequencing from B. rapa leaves and inflorescences. To analyze these data we developed a Reproducible Epigenomic Analysis pipeline using Galaxy and Jupyter, packaged into Docker images to facilitate transparency and reuse. We found that H3K27me3 covers roughly one-third of all B. rapa protein-coding genes and its presence correlates with low transcript levels. The comparative analysis between leaves and inflorescences suggested that the expression of various floral regulatory genes during development depends on H3K27me3. To demonstrate the importance of H3K27me3 for B. rapa development, we characterized a mutant line deficient in the H3K27 methyltransferase activity. We found that braA.clf mutant plants presented pleiotropic alterations, e.g., curly leaves due to increased expression and reduced H3K27me3 levels at AGAMOUS-like loci.ConclusionsWe characterized the epigenetic mark H3K27me3 at genome-wide levels and provide genetic evidence for its relevance in B. rapa development. Our work reveals the epigenomic landscape of H3K27me3 in B. rapa and provides novel genomics datasets and bioinformatics analytical resources. We anticipate that this work will lead the way to further epigenomic studies in the complex genome of Brassica crops.

Comprehensive comparative analysis of 5'-end RNA-sequencing methods.

RNA-Seq is an effective method to study the transcriptome, but specialized methods are required to identify 5’ ends of transcripts. Several published strategies exist for this specific purpose, but their relative merits have not been systematically analyzed. Here, we directly compare the performance of six such methods – testing five with cellular RNA as well as a novel spike-in RNA assay that helps address interpretation challenges that arise from uncertainties in annotation or RNA processing. Using a single human RNA sample, we constructed and sequenced 18 libraries with these methods and one standard, control RNA-Seq library. We find that the CAGE method performed best for mRNA and that most of its unannotated peaks are supported by evidence from other genomic methods. We then applied CAGE to eight brain-related samples and revealed sample-specific transcription start site (TSS) usage as well as a transcriptome-wide shift in TSS usage between fetal and adult brain.

Alternative 3′ UTRs Modify the Localization, Regulatory Potential, Stability, and Plasticity of mRNAs in Neuronal Compartments

Neurons localize mRNAs near synapses where their translation can be regulated by synaptic demand and activity. Differences in the 3' UTRs of mRNAs can change their localization, stability, and translational regulation. Using 3' end RNA sequencing of microdissected rat brain slices, we discovered a huge diversity in mRNA 3' UTRs, with many transcripts showing enrichment for a particular 3' UTR isoform in either somata or the neuropil. The 3' UTR isoforms of localized transcripts are significantly longer than the 3' UTRs of non-localized transcripts and often code for proteins associated with axons, dendrites, and synapses. Surprisingly, long 3' UTRs add not only new, but also duplicate regulatory elements. The neuropil-enriched 3' UTR isoforms have significantly longer half-lives than somata-enriched isoforms. Finally, the 3' UTR isoforms can be significantly altered by enhanced activity. Most of the 3' UTR plasticity is transcription dependent, but intriguing examples of changes that are consistent with altered stability, trafficking between compartments, or local "remodeling" remain.

Refined annotation of the complete genome of the phytopathogenic and xanthan producing Xanthomonas campestris pv. campestris strain B100 based on RNA sequence data

Bioinformatics tools and gene expression data were applied to identify new genes and to enhance the accuracy in genomic feature predictions for Xanthomonas campestris pv. campestris (Xcc) B100, a pathogen of cruciferous plants and model strain for the biosynthesis of xanthan, a polysaccharide with a multitude of commercial applications as a thickening agent. Results from 5′-enriched end RNA sequencing (RNA-seq) and total transcriptome RNA-seq experiments were used for this purpose. Functional gene annotations were updated where new evidence had emerged and start codon predictions were enhanced for 153 protein-coding genes (CDS). In total, 32 novel CDS, and 176 novel RNA genes and features were predicted, among them 77 isogenes of the small non-coding RNA sX9. Furthermore, the RNA-seq data facilitated the identification of 848 operons that included a total of 2551 CDS besides 1667 CDS that were mono-cistronically expressed.

Identification of a Novel MPL Loss of Function Mutation in a Patient with Cyclic Thrombocytopenia and Characterization of This Syndrome

Introduction: Cyclic thrombocytopenia (CT) is a rare disease characterized by periodic fluctuations in platelet counts. The etiology of this disease has not been fully elucidated. Here, we present molecular characterization of CT in a 53-year-old male patient who has platelet counts ranging between 1 x 109/L to >400 x 109/L during a cycle period of 40 days which has occurred over several years.Methods: Blood transcriptome profiles and plasma cytokine levels were investigated in 24 samples that were sequentially collected every 3-4 days to cover two complete cycles. Total RNA was extracted after lysing red blood cells. 3SEQ (3′-end RNA sequencing for expression quantification) was performed for transcriptome profiling. SAMSeq quantitative analyses were conducted to identify differentially expressed genes. Three screening criteria were applied to this data set to identify a panel of exclusive platelet-specific genes. Plasma TPO (thrombopoietin)/testosterone/ estradiol/CD41a/CD42b levels were detected by ELISA. A total of 62 cytokines were screened using a luminex immunoassay. MPL (TPO receptor) gene was Sanger-sequenced using blood DNA and mRNA templates. DNA was extracted from the patient’s hair follicles to exam the presence of the identified MPL mutation, and in-silico predictions were conducted to evaluate the MPL mutation. The wild-type (WT) and c.1210G>A mutant MPL expression constructs were generated and transfected into Ba/F3 cells for stable MPL expression. TPO-stimulated, IL-3 independent growth of MPL-expressing Ba/F3 cells were assessed to determine the function of MPL proteins.Results: Plasma TPO levels cycled between 6 to 2745 pg/mL during the sampling period; TPO levels inversely mirrored the fluctuation of platelet counts and preceded the latter by 3-4 days. Additional plasma cytokines demonstrated either an inverse correlation (e.g. FasL, GM-CSF, and TNF-β, etc.), or well synchronized correlation (e.g. BDNF, PDGF-BB, and RANTES etc.), with platelet counts. The patient was negative for platelet autoantibodies. Transcriptome analysis revealed 977 genes with expression that positively correlated with platelet count changes. Unsupervised clustering stratified these genes into subgroups, including: i) platelet-specific genes, which precede platelet count changes by 3-4 days; ii) platelet-modulated genes, which follow platelet count changes and are regulated by coagulation factors (e.g. F2) and platelet-contained growth factors (e.g. PDGF-BB and EGF); and iii) neutrophil-specific genes, which precede platelet count changes by 7-10 days. Among platelet-specific genes, 34 genes were identified to have >50-fold induction over a cycle, and are present in the platelet transcriptome [Rowley JW, 2011]. A heat map of these exclusive platelet genes is shown in Figure 1A.A novel MPL c.1210G>A heterozygous mutation, which leads to a p.Gly404Arg substitution, was identified in both blood and hair follicle DNA samples of the patient. This substitution resides in a highly conserved site and is predicted to be deleterious. In addition, the mutation creates a novel splice site, which results in the insertion of a 36 bp intron fragment in approximately 15% of MPL transcripts. In vitro analysis confirmed that MPL c.1210G>A is a loss-of-function (LOF) variant. Ba/F3 cells that express WT MPL exhibited IL-3 independent growth in the presence of TPO; however, the Gly404Arg mutant failed to support TPO-stimulated growth (Figure 1B).Conclusion: We describe a patient with CT who exhibits fluctuations of multiple cytokines including an inverse correlation between TPO and platelet counts, and profound gene expression changes in neutrophil- and platelet-specific gene expressions which precede platelet count fluctuations. These data suggest that the cyclic megakaryopoiesis and thrombopoiesis underlies pathogenesis of the disease. Moreover, the identified novel LOF MPL allele may contribute to the disease pathogenesis. The biologic and molecular annotation of this case presents a unique opportunity to examine the transcriptional regulation of platelet homeostasis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

NOD-like receptor signaling and inflammasome-related pathways are highlighted in psoriatic epidermis.

Psoriatic skin differs distinctly from normal skin by its thickened epidermis. Most gene expression comparisons utilize full-thickness biopsies, with substantial amount of dermis. We assayed the transcriptomes of normal, lesional, and non-lesional psoriatic epidermis, sampled as split-thickness skin grafts, with 5′-end RNA sequencing. We found that psoriatic epidermis contains more mRNA per total RNA than controls, and took this into account in the bioinformatic analysis. The approach highlighted innate immunity-related pathways in psoriasis, including NOD-like receptor (NLR) signaling and inflammasome activation. We demonstrated that the NLR signaling genes NOD2, PYCARD, CARD6, and IFI16 are upregulated in psoriatic epidermis, and strengthened these findings by protein expression. Interestingly, PYCARD, the key component of the inflammasome, showed an altered expression pattern in the lesional epidermis. The profiling of non-lesional skin highlighted PSORS4 and mitochondrially encoded transcripts, suggesting that their gene expression is altered already before the development of lesions. Our data suggest that all components needed for the active inflammasome are present in the keratinocytes of psoriatic skin. The characterization of inflammasome pathways provides further opportunities for therapy. Complementing previous transcriptome studies, our approach gives deeper insight into the gene regulation in psoriatic epidermis.

Genome-Wide Polyadenylation Maps Reveal Dynamic mRNA 3'-End Formation in the Failing Human Heart.

Alternative cleavage and polyadenylation (APA) of mRNA represents a layer of gene regulation that to date has remained unexplored in the heart. This phenomenon may be relevant, as the positioning of the poly(A) tail in mRNAs influences the length of the 3'-untranslated region (UTR), a critical determinant of gene expression. To investigate whether the 3'UTR length is regulated by APA in the human heart and whether this changes in the failing heart. We used 3'end RNA sequencing (e3'-Seq) to directly measure global patterns of APA in healthy and failing human heart specimens. By monitoring polyadenylation profiles in these hearts, we identified disease-specific APA signatures in numerous genes. Interestingly, many of the genes with shortened 3'UTRs in heart failure were enriched for functional groups such as RNA binding, whereas genes with longer 3'UTRs were enriched for cytoskeletal organization and actin binding. RNA sequencing in a larger series of human hearts revealed that these APA candidates are often differentially expressed in failing hearts, with an inverse correlation between 3'UTR length and the level of gene expression. Protein levels of the APA regulator, poly(A)-binding protein nuclear-1 were substantially downregulated in failing hearts. We provide genome-wide, high-resolution polyadenylation maps of the human heart and show that the 3'end formation of mRNA is dynamic in heart failure, suggesting that APA-mediated 3'UTR length modulation represents an additional layer of gene regulation in failing hearts.

Clinically Relevant Molecular Subtypes in Leiomyosarcoma.

Leiomyosarcoma is a malignant neoplasm with smooth muscle differentiation. Little is known about its molecular heterogeneity and no targeted therapy currently exists for leiomyosarcoma. Recognition of different molecular subtypes is necessary to evaluate novel therapeutic options. In a previous study on 51 leiomyosarcomas, we identified three molecular subtypes in leiomyosarcoma. The current study was performed to determine whether the existence of these subtypes could be confirmed in independent cohorts. Ninety-nine cases of leiomyosarcoma were expression profiled with 3'end RNA-Sequencing (3SEQ). Consensus clustering was conducted to determine the optimal number of subtypes. We identified 3 leiomyosarcoma molecular subtypes and confirmed this finding by analyzing publically available data on 82 leiomyosarcoma from The Cancer Genome Atlas (TCGA). We identified two new formalin-fixed, paraffin-embedded tissue-compatible diagnostic immunohistochemical markers; LMOD1 for subtype I leiomyosarcoma and ARL4C for subtype II leiomyosarcoma. A leiomyosarcoma tissue microarray with known clinical outcome was used to show that subtype I leiomyosarcoma is associated with good outcome in extrauterine leiomyosarcoma while subtype II leiomyosarcoma is associated with poor prognosis in both uterine and extrauterine leiomyosarcoma. The leiomyosarcoma subtypes showed significant differences in expression levels for genes for which novel targeted therapies are being developed, suggesting that leiomyosarcoma subtypes may respond differentially to these targeted therapies. We confirm the existence of 3 molecular subtypes in leiomyosarcoma using two independent datasets and show that the different molecular subtypes are associated with distinct clinical outcomes. The findings offer an opportunity for treating leiomyosarcoma in a subtype-specific targeted approach.