Failure of human rhombic lip differentiation underlies medulloblastoma formation.

Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain1-4. Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage5-8. By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL9,10. However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage3,4. Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2, CBFA2T3, PRDM6, UTX and OTX2. CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens, and G4 MB transcriptionally resembles these progenitors but are stalled in developmental time. Knockdown of OTX2 in model systems relieves this differentiation blockade, which allows MB cells to spontaneously proceed along normal developmental differentiation trajectories. The specific nature of the split human RL, which is destined to generate most of the neurons in the human brain, and its high level of susceptible EOMES+KI67+ unipolar brush cell progenitor cells probably predisposes our species to the development of G4 MB.

The transcriptional landscape of Shh medulloblastoma.

Sonic hedgehog medulloblastoma encompasses a clinically and molecularly diverse group of cancers of the developing central nervous system. Here, we use unbiased sequencing of the transcriptome across a large cohort of 250 tumors to reveal differences among molecular subtypes of the disease, and demonstrate the previously unappreciated importance of non-coding RNA transcripts. We identify alterations within the cAMP dependent pathway (GNAS, PRKAR1A) which converge on GLI2 activity and show that 18% of tumors have a genetic event that directly targets the abundance and/or stability of MYCN. Furthermore, we discover an extensive network of fusions in focally amplified regions encompassing GLI2, and several loss-of-function fusions in tumor suppressor genes PTCH1, SUFU and NCOR1. Molecular convergence on a subset of genes by nucleotide variants, copy number aberrations, and gene fusions highlight the key roles of specific pathways in the pathogenesis of Sonic hedgehog medulloblastoma and open up opportunities for therapeutic intervention.

Alternative Splicing of Opioid Receptor Genes Shows a Conserved Pattern for 6TM Receptor Variants.

The opioid receptor (OPR) family comprises the mu-, delta-, and kappa-opioid, and nociceptin receptors that belong to the superfamily of 7-transmembrane spanning G protein-coupled receptors (GPCRs). The mu-opioid receptor is the main target for clinically used opioid analgesics, and its biology has been extensively studied. The N-terminally truncated 6TM receptors isoform produced through alternative splicing of the OPRM1 gene displays unique signaling and analgesic properties, but it is unclear if other OPRs have the same ability. In this study, we have built a comprehensive map of alternative splicing events that produce 6TM receptor variants in all the OPRs and demonstrated their evolutionary conservation. We then obtained evidence for their translation through ribosomal footprint analysis. We discovered that N-terminally truncated 6TM GPCRs are rare in the human genome and OPRs are overrepresented in this group. Finally, we also observed a significant enrichment of 6TM GPCR genes among genes associated with pain, psychiatric disorders, and addiction. Understanding the biology of 6TM receptors and leveraging this knowledge for drug development should pave the way for novel therapies.

Alternative Splicing of the Delta-Opioid Receptor Gene Suggests Existence of New Functional Isoforms.

The delta-opioid receptor (DOPr) participates in mediating the effects of opioid analgesics. However, no selective agonists have entered clinical care despite potential to ameliorate many neurological and psychiatric disorders. In an effort to address the drug development challenges, the functional contribution of receptor isoforms created by alternative splicing of the three-exonic coding gene, OPRD1, has been overlooked. We report that the gene is transcriptionally more diverse than previously demonstrated, producing novel protein isoforms in humans and mice. We provide support for the functional relevance of splice variants through context-dependent expression profiling (tissues, disease model) and conservation of the transcriptional landscape in closely related vertebrates. The conserved alternative transcriptional events have two distinct patterns. First, cassette exon inclusions between exons 1 and 2 interrupt the reading frame, producing truncated receptor fragments comprising only the first transmembrane (TM) domain, despite the lack of exact exon orthologues between distant species. Second, a novel promoter and transcriptional start site upstream of exon 2 produces a transcript of an N-terminally truncated 6TM isoform. However, a fundamental difference in the exonic landscaping as well as translation and translation products poses limits for modelling the human DOPr receptor system in mice.

Deep Sequencing Reveals Spatially Distributed Distinct Hot Spot Mutations in DICER1-Related Multinodular Goiter.

CONTEXT: Nontoxic multinodular goiter (MNG) occurs frequently, but its genetic etiology is not well established. Familial MNG and MNG occurring with ovarian Sertoli-Leydig cell tumor are associated with germline DICER1 mutations. We recently identified second somatic DICER1 ribonuclease (RNase) IIIb mutations in two MNGs. OBJECTIVE: The objective of the study was to investigate the occurrence of somatic DICER1 mutations and mutational clonality in MNG. PATIENTS: MNGs from 15 patients (10 with and five without germline DICER1 mutations) were selected based on tissue availability. DESIGN: Core biopsies/scrapings (n = 70) were obtained, sampling areas of follicular hyperplasia, hyperplasia within colloid pools, unremarkable thyroid parenchyma, and areas of thyroid parenchyma, not classified. After capture with a Fluidigm access array, the coding sequence of DICER1 was deep sequenced using DNA from each core/scraping. RESULTS: All germline DICER1-mutated cases were found to harbor at least one RNase III mutation. Specifically, we identified 12 individually distinct DICER1 RNase IIIb hot spot mutations in 32 of the follicular hyperplasia or hyperplasia within colloid pools cores/scrapings. These mutations are predicted to affect the metal-ion binding residues at positions p.Glu1705, p.Asp1709, p.Gly1809, p.Asp1810, and p.Glu1813. Somatic RNase IIIb mutations were identified in the 10 DICER1 germline mutated MNGs as follows: two cases contained one somatic mutation, five cases contained two mutations, and three cases contained three distinct somatic hot spot mutations. No RNase IIIb mutations were identified in the MNGs from individuals without germline DICER1 mutations. CONCLUSIONS: This study demonstrates that nodules within MNG occurring in DICER1 syndrome are associated with spatially distributed somatic DICER1 RNase IIIb mutations.

Fusion of TTYH1 with the C19MC microRNA cluster drives expression of a brain-specific DNMT3B isoform in the embryonal brain tumor ETMR.

Embryonal tumors with multilayered rosettes (ETMRs) are rare, deadly pediatric brain tumors characterized by high-level amplification of the microRNA cluster C19MC. We performed integrated genetic and epigenetic analyses of 12 ETMR samples and identified, in all cases, C19MC fusions to TTYH1 driving expression of the microRNAs. ETMR tumors, cell lines and xenografts showed a specific DNA methylation pattern distinct from those of other tumors and normal tissues. We detected extreme overexpression of a previously uncharacterized isoform of DNMT3B originating at an alternative promoter that is active only in the first weeks of neural tube development. Transcriptional and immunohistochemical analyses suggest that C19MC-dependent DNMT3B deregulation is mediated by RBL2, a known repressor of DNMT3B. Transfection with individual C19MC microRNAs resulted in DNMT3B upregulation and RBL2 downregulation in cultured cells. Our data suggest a potential oncogenic re-engagement of an early developmental program in ETMR via epigenetic alteration mediated by an embryonic, brain-specific DNMT3B isoform.