Heterogeneous contribution of microdeletions in the development of common generalised and focal epilepsies.

BACKGROUND: Microdeletions are known to confer risk to epilepsy, particularly at genomic rearrangement 'hotspot' loci. However, microdeletion burden not overlapping these regions or within different epilepsy subtypes has not been ascertained. OBJECTIVE: To decipher the role of microdeletions outside hotspots loci and risk assessment by epilepsy subtype. METHODS: We assessed the burden, frequency and genomic content of rare, large microdeletions found in a previously published cohort of 1366 patients with genetic generalised epilepsy (GGE) in addition to two sets of additional unpublished genome-wide microdeletions found in 281 patients with rolandic epilepsy (RE) and 807 patients with adult focal epilepsy (AFE), totalling 2454 cases. Microdeletions were assessed in a combined and subtype-specific approaches against 6746 controls. RESULTS: When hotspots are considered, we detected an enrichment of microdeletions in the combined epilepsy analysis (adjusted p=1.06×10-6,OR 1.89, 95% CI 1.51 to 2.35). Epilepsy subtype-specific analyses showed that hotspot microdeletions in the GGE subgroup contribute most of the overall signal (adjusted p=9.79×10-12, OR 7.45, 95% CI 4.20-13.5). Outside hotspots , microdeletions were enriched in the GGE cohort for neurodevelopmental genes (adjusted p=9.13×10-3,OR 2.85, 95% CI 1.62-4.94). No additional signal was observed for RE and AFE. Still, gene-content analysis identified known (NRXN1, RBFOX1 and PCDH7) and novel (LOC102723362) candidate genes across epilepsy subtypes that were not deleted in controls. CONCLUSIONS: Our results show a heterogeneous effect of recurrent and non-recurrent microdeletions as part of the genetic architecture of GGE and a minor contribution in the aetiology of RE and AFE.

Wnt signaling induces transcription, spatial proximity, and translocation of fusion gene partners in human hematopoietic cells.

Chromosomal translocations are frequently associated with a wide variety of cancers, particularly hematologic malignancies. A recurrent chromosomal abnormality in acute myeloid leukemia is the reciprocal translocation t(8;21) that fuses RUNX1 and ETO genes. We report here that Wnt/ß-catenin signaling increases the expression of ETO and RUNX1 genes in human hematopoietic progenitors. We found that ß-catenin is rapidly recruited into RNA polymerase II transcription factories (RNAPII-Ser5) and that ETO and RUNX1 genes are brought into close spatial proximity upon Wnt3a induction. Notably, long-term treatment of cells with Wnt3a induces the generation a frequent RUNX1-ETO translocation event. Thus, Wnt/ß-catenin signaling induces transcription and translocation of RUNX1 and ETO fusion gene partners, opening a novel window to understand the onset/development of leukemia.

Alternative RUNX1 Promoter Regulation by Wnt/ß-Catenin Signaling in Leukemia Cells and Human Hematopoietic Progenitors.

Two distantly located promoter regions regulate the dynamic expression of RUNX genes during development: distal P1 and proximal P2 promoters. We have recently described that ß-catenin increases total Runx1 mRNA levels in human CD34(+) hematopoietic progenitors and enhances spatial proximity with its translocation partner ETO. Here, we report that induction of Wnt/ß-catenin signaling in HL60 and Jurkat leukemia-derived cell lines and CD34(+) progenitors selectively activate the production of the longer distal P1-Runx1 mRNA isoform. Gain- and loss-of-function experiments revealed that the differential increase in P1-Runx1 expression is accomplished through a minimal ß-catenin responsive region that includes a highly conserved TCF/LEF-binding element, located -20/-16 bp upstream of the canonical distal P1-Runx1 transcription start site. We conclude that the distal P1-Runx1 promoter is a direct transcriptional target of Wnt/ß-catenin signaling that may be important in normal hematopoiesis or its transition into malignant stem cells during the onset or progression of leukemia.

Synaptic Wnt/GSK3ß Signaling Hub in Autism.

Hundreds of genes have been associated with autism spectrum disorders (ASDs) and the interaction of weak and de novo variants derive from distinct autistic phenotypes thus making up the "spectrum." The convergence of these variants in networks of genes associated with synaptic function warrants the study of cell signaling pathways involved in the regulation of the synapse. The Wnt/ß-catenin signaling pathway plays a central role in the development and regulation of the central nervous system and several genes belonging to the cascade have been genetically associated with ASDs. In the present paper, we review basic information regarding the role of Wnt/ß-catenin signaling in excitatory/inhibitory balance (E/I balance) through the regulation of pre- and postsynaptic compartments. Furthermore, we integrate information supporting the role of the glycogen synthase kinase 3ß (GSK3ß) in the onset/development of ASDs through direct modulation of Wnt/ß-catenin signaling. Finally, given GSK3ß activity as key modulator of synaptic plasticity, we explore the potential of this kinase as a therapeutic target for ASD.

Early Transcriptional Changes Induced by Wnt/ß-Catenin Signaling in Hippocampal Neurons.

Wnt/ß-catenin signaling modulates brain development and function and its deregulation underlies pathological changes occurring in neurodegenerative and neurodevelopmental disorders. Since one of the main effects of Wnt/ß-catenin signaling is the modulation of target genes, in the present work we examined global transcriptional changes induced by short-term Wnt3a treatment (4 h) in primary cultures of rat hippocampal neurons. RNAseq experiments allowed the identification of 170 differentially expressed genes, including known Wnt/ß-catenin target genes such as Notum, Axin2, and Lef1, as well as novel potential candidates Fam84a, Stk32a, and Itga9. Main biological processes enriched with differentially expressed genes included neural precursor (GO:0061364, p-adjusted = 2.5 × 10-7), forebrain development (GO:0030900, p-adjusted = 7.3 × 10-7), and stem cell differentiation (GO:0048863 p-adjusted = 7.3 × 10-7). Likewise, following activation of the signaling cascade, the expression of a significant number of genes with transcription factor activity (GO:0043565, p-adjusted = 4.1 × 10-6) was induced. We also studied molecular networks enriched upon Wnt3a activation and detected three highly significant expression modules involved in glycerolipid metabolic process (GO:0046486, p-adjusted = 4.5 × 10-19), learning or memory (GO:0007611, p-adjusted = 4.0 × 10-5), and neurotransmitter secretion (GO:0007269, p-adjusted = 5.3 × 10-12). Our results indicate that Wnt/ß-catenin mediated transcription controls multiple biological processes related to neuronal structure and activity that are affected in synaptic dysfunction disorders.

Enhanced CRAd activity using enhancer motifs driven by a nucleosome positioning sequence.

Cancer development involves changes driven by the epigenetic machinery, including nucleosome positioning. Recently, the concept that adenoviral replication may be driven by tumor specific promoters (TSPs) gained support, and several conditionally replicative adenoviruses (CRAd) exhibited therapeutic efficacy in clinical trials. Here, we show for the first time that placing a nucleosome positioning sequence (NPS) upstream of a TSP combined with Wnt-responsive motifs (pART enhancer) enhanced the TSP transcriptional activity and increased the lytic activity of a CRAd. pART enhanced the transcriptional activity of the gastrointestinal cancer (GIC)-specific REG1A promoter (REG1A-pr); moreover, pART also increased the in vitro lytic activity of a CRAd whose replication was driven by REG1A-Pr. The pART enhancer effect in vitro and in vivo was strictly dependent on the presence of the NPS. Indeed, deletion of the NPS was strongly deleterious for the in vivo antitumor efficacy of the CRAd on orthotopically established pancreatic xenografts. pART also enhanced the specific activity of other heterologous promoters; moreover, the NPS was also able to enhance the responsiveness of hypoxia- and NFκB-response elements. We conclude that NPS could be useful for gene therapy approaches in cancer as well as other diseases.

Canonical Wnt3a modulates intracellular calcium and enhances excitatory neurotransmission in hippocampal neurons.

A role for Wnt signal transduction in the development and maintenance of brain structures is widely acknowledged. Recent studies have suggested that Wnt signaling may be essential for synaptic plasticity and neurotransmission. However, the direct effect of a Wnt protein on synaptic transmission had not been demonstrated. Here we show that nanomolar concentrations of purified Wnt3a protein rapidly increase the frequency of miniature excitatory synaptic currents in embryonic rat hippocampal neurons through a mechanism involving a fast influx of calcium from the extracellular space, induction of post-translational modifications on the machinery involved in vesicle exocytosis in the presynaptic terminal leading to spontaneous Ca(2+) transients. Our results identify the Wnt3a protein and a member of its complex receptor at the membrane, the low density lipoprotein receptor-related protein 6 (LRP6) coreceptor, as key molecules in neurotransmission modulation and suggest cross-talk between canonical and Wnt/Ca(2+) signaling in central neurons.

Biphasic effects of copper on neurotransmission in rat hippocampal neurons.

The importance of copper in the CNS is well documented, but the mechanisms related to its brain functions are poorly understood. Copper is released at the synaptic cleft, where it may modulate neurotransmission. To understand the functional impact of copper on the neuronal network, we have analyzed the synaptic activity of primary rat hippocampal neurons by using different approaches including whole cell patch clamp, recording of calcium transients, immunofluorescence and western blot. Here, we show that copper produces biphasic changes in neurotransmission. When copper is acutely applied to the plate it blocks neurotransmission. Interestingly, when it is applied for 3 h to hippocampal neurons it mainly increases the frequency and amplitude of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)ergic currents (control: 0.21 ± 0.05 Hz/22.9 ± 1.3 pA; copper: 0.68 ± 0.16 Hz/30.5 ± 2.5 pA), intracellular calcium transients (control: 0.05 ± 0.013 Hz; copper: 0.11 ± 0.02 Hz) and evoked AMPA currents (control: EC50 8.3 ± 0.5 µM; copper: EC50 2.9 ± 0.2 µM). Moreover, our results suggest that copper increases GluA1 subunit levels of the AMPA receptor through the anchorage of AMPA receptors to the plasma membrane as a result of PSD-95 accumulation. We also found that copper-treated neurons displayed an undistinguishable neurotransmission to control neurons after 24 h of treatment, indicating that changes in neurotransmission induced by copper at 3 h of incubation are homeostatically regulated after long-term exposure to the metal. Together, our data reveal an unexpected biphasic effect of copper on neurotransmission, which may be relevant to understand the effects of this ion in brain diseases that display copper dyshomeostasis such as that observed in Alzheimer's disease (AD).

Wnt/ß-Catenin-Dependent Transcription in Autism Spectrum Disorders.

Autism spectrum disorders (ASD) is a heterogeneous group of neurodevelopmental disorders characterized by synaptic dysfunction and defects in dendritic spine morphology. In the past decade, an extensive list of genes associated with ASD has been identified by genome-wide sequencing initiatives. Several of these genes functionally converge in the regulation of the Wnt/ß-catenin signaling pathway, a conserved cascade essential for stem cell pluripotency and cell fate decisions during development. Here, we review current information regarding the transcriptional program of Wnt/ß-catenin signaling in ASD. First, we discuss that Wnt/ß-catenin gain and loss of function studies recapitulate brain developmental abnormalities associated with ASD. Second, transcriptomic approaches using patient-derived induced pluripotent stem cells (iPSC) cells, featuring mutations in high confidence ASD genes, reveal a significant dysregulation in the expression of Wnt signaling components. Finally, we focus on the activity of chromatin-remodeling proteins and transcription factors considered high confidence ASD genes, including CHD8, ARID1B, ADNP, and TBR1, that regulate Wnt/ß-catenin-dependent transcriptional activity in multiple cell types, including pyramidal neurons, interneurons and oligodendrocytes, cells which are becoming increasingly relevant in the study of ASD. We conclude that the level of Wnt/ß-catenin signaling activation could explain the high phenotypical heterogeneity of ASD and be instrumental in the development of new diagnostics tools and therapies.

Copy number variants in lipid metabolism genes are associated with gallstones disease in men.

Gallstones Disease (GSD) is one of the most common digestive diseases requiring hospitalization and surgical procedures in the world. GSD has a high prevalence in populations with European or Amerindian ancestry (10-20%) and the influence of genetic factors is broadly acknowledged. However, known genetic variants do not entirely explain the disease heritability suggesting that additional genetic variants remain to be identified. Here, we examined the association of copy number variants (CNVs) with GSD in a sample of 4778 individuals (1929 GSD cases and 2849 controls) including two European cohorts from Germany (n = 3702) and one admixed Latin American cohort from Chile (n = 1076). We detected 2936 large and rare CNVs events (size > 100 kb, frequency < 1%). Case-control burden analysis and generalized linear regression models revealed significant association of CNVs with GSD in men, with the strongest effect observed with CNVs overlapping lipid metabolism genes (p-value = 6.54 × 10-4; OR = 2.76; CI 95% = 1.53-4.89). Our results indicate a clear link between CNVs and GSD in men and provides additional evidence that the genetic components of risk for GSD are complex, can be sex specific and include CNVs affecting genes involved in lipid metabolism.

Variants in ABCG8 and TRAF3 genes confer risk for gallstone disease in admixed Latinos with Mapuche Native American ancestry.

Latin Americans and Chilean Amerindians have the highest prevalence of gallstone disease (GSD) and gallbladder cancer (GBC) in the world. A handful of loci have been associated with GSD in populations of predominantly European ancestry, however, they only explain a small portion of the genetic component of the disease. Here, we performed a genome-wide association study (GWAS) for GSD in 1,095 admixed Chilean Latinos with Mapuche Native American ancestry. Disease status was assessed by cholecystectomy or abdominal ultrasonography. Top-10 candidate variants surpassing the suggestive cutoff of P < 1 × 10-5 in the discovery cohort were genotyped in an independent replication sample composed of 1,643 individuals. Variants with positive replication were further examined in two European GSD populations and a Chilean GBC cohort. We consistently replicated the association of ABCG8 gene with GSD (rs11887534, P = 3.24 × 10-8, OR = 1.74) and identified TRAF3 (rs12882491, P = 1.11 × 10-7, OR = 1.40) as a novel candidate gene for the disease in admixed Chilean Latinos. ABCG8 and TRAF3 variants also conferred risk to GBC. Gene expression analyses indicated that TRAF3 was significantly decreased in gallbladder (P = 0.015) and duodenal mucosa (P = 0.001) of GSD individuals compared to healthy controls, where according to GTEx data in the small intestine, the presence of the risk allele contributes to the observed effect. We conclude that ABCG8 and TRAF3 genes are associated with GSD and GBC in admixed Latinos and that decreased TRAF3 levels could enhance gallbladder inflammation as is observed in GSD and GSD-associated GBC.

Whole Genome Sequence, Variant Discovery and Annotation in Mapuche-Huilliche Native South Americans.

Whole human genome sequencing initiatives help us understand population history and the basis of genetic diseases. Current data mostly focuses on Old World populations, and the information of the genomic structure of Native Americans, especially those from the Southern Cone is scant. Here we present annotation and variant discovery from high-quality complete genome sequences of a cohort of 11 Mapuche-Huilliche individuals (HUI) from Southern Chile. We found approximately 3.1 × 106 single nucleotide variants (SNVs) per individual and identified 403,383 (6.9%) of novel SNVs events. Analyses of large-scale genomic events detected 680 copy number variants (CNVs) and 4,514 structural variants (SVs), including 398 and 1,910 novel events, respectively. Global ancestry composition of HUI genomes revealed that the cohort represents a sample from a marginally admixed population from the Southern Cone, whose main genetic component derives from Native American ancestors. Additionally, we found that HUI genomes contain variants in genes associated with 5 of the 6 leading causes of noncommunicable diseases in Chile, which may have an impact on the risk of prevalent diseases in Chilean and Amerindian populations. Our data represents a useful resource that can contribute to population-based studies and for the design of early diagnostics or prevention tools for Native and admixed Latin American populations.

Wnt/ß-catenin signaling stimulates the expression and synaptic clustering of the autism-associated Neuroligin 3 gene.

Synaptic abnormalities have been described in individuals with autism spectrum disorders (ASD). The cell-adhesion molecule Neuroligin-3 (Nlgn3) has an essential role in the function and maturation of synapses and NLGN3 ASD-associated mutations disrupt hippocampal and cortical function. Here we show that Wnt/ß-catenin signaling increases Nlgn3 mRNA and protein levels in HT22 mouse hippocampal cells and primary cultures of rat hippocampal neurons. We characterized the activity of mouse and rat Nlgn3 promoter constructs containing conserved putative T-cell factor/lymphoid enhancing factor (TCF/LEF)-binding elements (TBE) and found that their activity is significantly augmented in Wnt/ß-catenin cell reporter assays. Chromatin immunoprecipitation (ChIP) assays and site-directed mutagenesis experiments revealed that endogenous ß-catenin binds to novel TBE consensus sequences in the Nlgn3 promoter. Moreover, activation of the signaling cascade increased Nlgn3 clustering and co- localization with the scaffold PSD-95 protein in dendritic processes of primary neurons. Our results directly link Wnt/ß-catenin signaling to the transcription of the Nlgn3 gene and support a functional role for the signaling pathway in the dysregulation of excitatory/inhibitory neuronal activity, as is observed in animal models of ASD.

Duplications at 19q13.33 in patients with neurodevelopmental disorders.

OBJECTIVE: After the recent publication of the first patients with disease-associated missense variants in the GRIN2D gene, we evaluate the effect of copy number variants (CNVs) overlapping this gene toward the presentation of neurodevelopmental disorders (NDDs). METHODS: We explored ClinVar (number of CNVs = 50,794) and DECIPHER (number of CNVs = 28,085) clinical databases of genomic variations for patients with copy number changes overlapping the GRIN2D gene at the 19q13.33 locus and evaluated their respective phenotype alongside their frequency, gene content, and expression, with publicly available reference databases. RESULTS: We identified 11 patients with microduplications at the 19q13.33 locus. The majority of CNVs arose de novo, and comparable CNVs are not present in control databases. All patients were reported to have NDDs and dysmorphic features as the most common clinical phenotype (N = 8/11), followed by seizures (N = 6/11) and intellectual disability (N = 5/11). All duplications shared a consensus region of 405 kb overlapping 13 genes. After screening for duplication tolerance in control populations, positive gene brain expression, and gene dosage sensitivity analysis, we highlight 4 genes for future evaluation: CARD8, C19orf68, KDELR1, and GRIN2D, which are promising candidates for disease causality. Furthermore, investigation of the literature especially supports GRIN2D as the best candidate gene. CONCLUSIONS: Our study presents dup19q13.33 as a novel duplication syndrome locus associated with NDDs. CARD8, C19orf68, KDELR1, and GRIN2D are promising candidates for functional follow-up.

The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state.

With about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients.

Wnt signaling involvement in beta-amyloid-dependent neurodegeneration.

Alzheimer's disease (AD) is a progressive dementia paralleled by selective neuronal death, which is probably caused by the cytotoxic effects of the amyloid-beta peptide (Abeta). We have observed that Abeta-dependent neurotoxicity induces a loss of function of Wnt signaling components and that activation of this signaling cascade prevent such cytotoxic effects. Therefore we propose that compounds which mimic this signaling cascade may be candidates for therapeutic intervention in Alzheimer's patients.

Wnt/ß-catenin signaling in Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disorder that causes a progressive decline of mental and cognitive processes such as memory, judgment and reasoning. We proposed earlier that a sustained loss of function of Wnt/ß- catenin signaling components underlies the onset and progression of the disease. Here, we discuss recent data on the involvement of Wnt/b-catenin signaling on amyloid precursor protein (APP) processing, Aß peptide neurotoxicity, τ phosphorylation, and modulation of Apolipoprotein E function in the brain. We conclude that several components of the cascade are actively engaged in the events leading to AD neuropathology and propose that compounds that mimic activation of this signaling cascade, such as lithium, should be considered for therapeutic intervention in Alzheimer's patients. In summary, data accumulated during the past decade confirm some important predictions of our hypothesis where components of this signaling cascade are actively engaged in the events leading to AD neuropathology and that compounds that mimic activation of this signaling cascade, such as lithium, should be considered for therapeutic intervention in Alzheimer's patients.

Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells.

Cumulative evidence indicates that Wnt pathways play crucial and diverse roles to assemble the neuromuscular junction (NMJ), a peripheral synapse characterized by the clustering of acetylcholine receptors (AChR) on postsynaptic densities. The molecular determinants of Wnt effects at the NMJ are still to be fully elucidated. We report here that the Wnt receptor Frizzled-9 (Fzd9) is expressed in developing skeletal muscles during NMJ synaptogenesis. In cultured myotubes, gain- and loss-of-function experiments revealed that Fzd9-mediated signaling impairs the AChR-clustering activity of agrin, an organizer of postsynaptic differentiation. Overexpression of Fzd9 induced the cytosolic accumulation of ß-catenin, a key regulator of Wnt signaling. Consistently, Fzd9 and ß-catenin localize in the postsynaptic domain of embryonic NMJs in vivo. Our findings represent the first evidence pointing to a crucial role of a Fzd-mediated, ß-catenin-dependent signaling on the assembly of the vertebrate NMJ.

Overrepresentation of glutamate signaling in Alzheimer's disease: network-based pathway enrichment using meta-analysis of genome-wide association studies.

Genome-wide association studies (GWAS) have successfully identified several risk loci for Alzheimer's disease (AD). Nonetheless, these loci do not explain the entire susceptibility of the disease, suggesting that other genetic contributions remain to be identified. Here, we performed a meta-analysis combining data of 4,569 individuals (2,540 cases and 2,029 healthy controls) derived from three publicly available GWAS in AD and replicated a broad genomic region (>248,000 bp) associated with the disease near the APOE/TOMM40 locus in chromosome 19. To detect minor effect size contributions that could help to explain the remaining genetic risk, we conducted network-based pathway analyses either by extracting gene-wise p-values (GW), defined as the single strongest association signal within a gene, or calculated a more stringent gene-based association p-value using the extended Simes (GATES) procedure. Comparison of these strategies revealed that ontological sub-networks (SNs) involved in glutamate signaling were significantly overrepresented in AD (p<2.7×10(-11), p<1.9×10(-11); GW and GATES, respectively). Notably, glutamate signaling SNs were also found to be significantly overrepresented (p<5.1×10(-8)) in the Alzheimer's disease Neuroimaging Initiative (ADNI) study, which was used as a targeted replication sample. Interestingly, components of the glutamate signaling SNs are coordinately expressed in disease-related tissues, which are tightly related to known pathological hallmarks of AD. Our findings suggest that genetic variation within glutamate signaling contributes to the remaining genetic risk of AD and support the notion that functional biological networks should be targeted in future therapies aimed to prevent or treat this devastating neurological disorder.