This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

Glioblastoma are aggressive astrocytic brain tumours characterized by microvascular proliferation and an abnormal vasculature, giving rise to brain oedema and increased patient morbidity. Here, we have characterized the transcriptome of tumour-associated blood vessels and describe a gene signature clearly associated with pleomorphic, pathologically altered vessels in human glioblastoma (grade IV glioma). We identified 95 genes differentially expressed in glioblastoma vessels, while no significant differences in gene expression were detected between vessels in non-malignant brain and grade II glioma. Differential vascular expression of ANGPT2, CD93, ESM1, ELTD1, FILIP1L and TENC1 in human glioblastoma was validated by immunohistochemistry, using a tissue microarray. Through qPCR analysis of gene induction in primary endothelial cells, we provide evidence that increased VEGF-A and TGFβ2 signalling in the tumour microenvironment is sufficient to invoke many of the changes in gene expression noted in glioblastoma vessels. Notably, we found an enrichment of Smad target genes within the distinct gene signature of glioblastoma vessels and a significant increase of Smad signalling complexes in the vasculature of human glioblastoma in situ. This indicates a key role of TGFβ signalling in regulating vascular phenotype and suggests that, in addition to VEGF-A, TGFβ2 may represent a new target for vascular normalization therapy.

BackgroundSince different types of genetic variants, from single nucleotide variants (SNVs) to large chromosomal rearrangements, underlie intellectual disability, we evaluated the use of whole-genome sequencing (WGS) rather than chromosomal microarray analysis (CMA) as a first-line genetic diagnostic test.MethodsWe analyzed three cohorts with short-read WGS: (i) a retrospective cohort with validated copy number variants (CNVs) (cohort 1, n = 68), (ii) individuals referred for monogenic multi-gene panels (cohort 2, n = 156), and (iii) 100 prospective, consecutive cases referred to our center for CMA (cohort 3). Bioinformatic tools developed include FindSV, SVDB, Rhocall, Rhoviz, and vcf2cytosure.ResultsFirst, we validated our structural variant (SV)-calling pipeline on cohort 1, consisting of three trisomies and 79 deletions and duplications with a median size of 850 kb (min 500 bp, max 155 Mb). All variants were detected. Second, we utilized the same pipeline in cohort 2 and analyzed with monogenic WGS panels, increasing the diagnostic yield to 8%. Next, cohort 3 was analyzed by both CMA and WGS. The WGS data was processed for large (> 10 kb) SVs genome-wide and for exonic SVs and SNVs in a panel of 887 genes linked to intellectual disability as well as genes matched to patient-specific Human Phenotype Ontology (HPO) phenotypes. This yielded a total of 25 pathogenic variants (SNVs or SVs), of which 12 were detected by CMA as well. We also applied short tandem repeat (STR) expansion detection and discovered one pathologic expansion in ATXN7. Finally, a case of Prader-Willi syndrome with uniparental disomy (UPD) was validated in the WGS data.Important positional information was obtained in all cohorts. Remarkably, 7% of the analyzed cases harbored complex structural variants, as exemplified by a ring chromosome and two duplications found to be an insertional translocation and part of a cryptic unbalanced translocation, respectively.ConclusionThe overall diagnostic rate of 27% was more than doubled compared to clinical microarray (12%). Using WGS, we detected a wide range of SVs with high accuracy. Since the WGS data also allowed for analysis of SNVs, UPD, and STRs, it represents a powerful comprehensive genetic test in a clinical diagnostic laboratory setting.

Deregulation of microRNA (miRNA) expression has been documented in diffuse large B-cell lymphoma (DLBCL). However, the impact of miRNAs and their machinery in DLBCL is not fully determined. Here, we assessed the role of miRNA expression and their processing genes in DLBCL development. Using microarray and RT-qPCR approaches, we quantified global miRNAs and core components of miRNA-processing genes expression in 75 DLBCLs (56 de novo and 19 transformed) and 10 lymph nodes (LN). Differential miRNA signatures were identified between DLBCLs and LNs, or between the de novo and transformed DLBCLs. We also identified subsets of miRNAs associated with germinal center B-cell phenotype, BCL6 and IRF4 expression, and clinical staging. In addition, we showed a significant over-expression of TARBP2 in de novo DLBCLs as compared with LNs, and decreased expression of DROSHA, DICER, TARBP2 and PACT in transformed as compared with de novo cases. Interestingly, cases with high TARBP2 and DROSHA expression had a poorer chemotherapy response. We further showed that TARBP2 can regulate miRNA-processing efficiency in DLBCLs, and its expression inhibition decreases cell growth and increases apoptosis in DLBCL cell lines. Our findings provide new insights for the understanding of miRNAs and its machinery in DLBCL.

Human multiple myeloma (MM) represents a highly aneuploid tumor as shown by cytogenetic studies. This may partly explain the heterogeneity with regard to growth factor requirements demonstrated among MM cells. We have previously reported the expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) mRNA in some MM cell lines. In this study we investigated the role of IGF-I as a growth and/or survival factor in three MM cell lines: LP-1, EJM, and Karpas 707. We report that all cell lines expressed IGF-I and IGF-IR mRNA and protein. LP-1 and Karpas 707, but not EJM, were stimulated to proliferation in a dose-dependent manner by exogenous IGF-I. An IGF-IR blocking antibody inhibited both the IGF-I-induced and spontaneous growth of LP-1, and Karpas 707, while the EJM cell line was unaffected by the addition of the antibody. In conclusion, our results show that IGF-I can act as a growth factor in human MM, and they suggest that an autocrine IGF-I loop may contribute to the growth and survival in some MM cell lines.