15q Duplication Research Articles

Transcriptomic and proteomic profiling of glial versus neuronal Dube3a overexpression reveals common molecular changes in gliopathic epilepsies

Epilepsy affects millions of individuals worldwide and many cases are pharmacoresistant. Duplication 15q syndrome (Dup15q) is a genetic disorder caused by duplications of the 15q11.2-q13.1 region. Phenotypes include a high rate of pharmacoresistant epilepsy. We developed a Dup15q model in Drosophila melanogaster that recapitulates seizures in Dup15q by over-expressing fly Dube3a or human UBE3A in glial cells, but not neurons, implicating glia in the Dup15q epilepsy phenotype. We compared Dube3a overexpression in glia (repo>Dube3a) versus neurons (elav>Dube3a) using transcriptomics and proteomics of whole fly head extracts. We identified 851 transcripts differentially regulated in repo>Dube3a, including an upregulation of glutathione S-transferase (GST) genes that occurred cell autonomously within glial cells. We reliably measured approximately 2,500 proteins by proteomics, most of which were also quantified at the transcript level. Combined transcriptomic and proteomic analysis revealed an enrichment of 21 synaptic transmission genes downregulated at the transcript and protein in repo>Dube3a indicating synaptic proteins change in a cell non-autonomous manner in repo>Dube3a flies. We identified 6 additional glia originating bang-sensitive seizure lines and found upregulation of GSTs in 4 out of these 6 lines. These data suggest GST upregulation is common among gliopathic seizures and may ultimately provide insight for treating epilepsy.

An Unbiased Drug Screen for Seizure Suppressors in Duplication 15q Syndrome Reveals 5-HT1A and Dopamine Pathway Activation as Potential Therapies

BackgroundDuplication 15q (Dup15q) syndrome is a rare neurogenetic disorder characterized by autism and pharmacoresistant epilepsy. Most individuals with isodicentric duplications have been on multiple medications to control seizures. We recently developed a model of Dup15q in Drosophila by elevating levels of fly Dube3a in glial cells using repo-GAL4, not neurons. In contrast to other Dup15q models, these flies develop seizures that worsen with age. MethodsWe screened repo>Dube3a flies for approved compounds that can suppress seizures. Flies 3 to 5 days old were exposed to compounds in the fly food during development. Flies were tested using a bang sensitivity assay for seizure recovery time. At least 40 animals were tested per experiment, with separate testing for male and female flies. Studies of K+ content in glial cells of the fly brain were also performed using a fluorescent K+ indicator. ResultsWe identified 17 of 1280 compounds in the Prestwick Chemical Library that could suppress seizures. Eight compounds were validated in secondary screening. Four of these compounds regulated either serotonergic or dopaminergic signaling, and subsequent experiments confirmed that seizure suppression occurred primarily through stimulation of serotonin receptor 5-HT1A. Additional studies of K+ levels showed that Dube3a regulation of the Na+/K+ exchanger ATPα (adenosine triphosphatase α) in glia may be modulated by serotonin/dopamine signaling, causing seizure suppression. ConclusionsBased on these pharmacological and genetic studies, we present an argument for the use of 5-HT1A agonists in the treatment of Dup15q epilepsy.

Quantitative Gait Analysis in Duplication 15q Syndrome and Nonsyndromic ASD.

Motor impairments occur frequently in genetic syndromes highly penetrant for autism spectrum disorder (syndromic ASD) and in individuals with ASD without a genetic diagnosis (nonsyndromic ASD). In particular, abnormalities in gait in ASD have been linked to language delay, ASD severity, and likelihood of having a genetic disorder. Quantitative measures of motor function can improve our ability to evaluate motor differences in individuals with syndromic and nonsyndromic ASD with varying levels of intellectual disability and adaptive skills. To evaluate this methodology, we chose to use quantitative gait analysis to study duplication 15q syndrome (dup15q syndrome), a genetic disorder highly penetrant for motor delays, intellectual disability, and ASD. We evaluated quantitative gait variables in individuals with dup15q syndrome (n = 39) and nonsyndromic ASD (n = 21) and compared these data to a reference typically developing cohort. We found a gait pattern of slow pace, poor postural control, and large gait variability in dup15q syndrome. Our findings improve characterization of motor function in dup15q syndrome and nonsyndromic ASD. Quantitative gait analysis can be used as a translational method and can improve our identification of clinical endpoints to be used in treatment trials for these syndromes. Autism Res 2020, 13: 1102-1110. © 2020 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Motor impairments, particularly abnormalities in walking, occur frequently in genetic syndromes highly penetrant for autism spectrum disorder (syndromic ASD). Here, using quantitative gait analysis, we find that individuals with duplication 15q syndrome have an atypical gait pattern that differentiates them from typically developing and nonsyndromic ASD individuals. Our findings improve motor characterization in dup15q syndrome and nonsyndromic ASD.

Genome and RNA sequencing in patients with methylmalonic aciduria of unknown cause

PurposeOur laboratory has classified patients with methylmalonic aciduria using somatic cell studies for over four decades. We have accumulated 127 fibroblast lines from patients with persistent elevated methylmalonic acid (MMA) levels in which no genetic cause could be identified. Cultured fibroblasts from 26 of these patients had low [14C]propionate incorporation into macromolecules, possibly reflecting decreased methylmalonyl-CoA mutase function. MethodsGenome sequencing (GS), copy-number variation (CNV) analysis, and RNA sequencing were performed on genomic DNA and complementary DNA (cDNA) from these 26 patients. ResultsNo patient had two pathogenic variants in any gene associated with cobalamin metabolism. Nine patients had heterozygous variants of unknown significance previously identified by a next-generation sequencing (NGS) panel targeting cobalamin metabolic genes. Three patients had pathogenic changes in genes not associated with cobalamin metabolism (PCCA, EPCAM, and a 17q12 duplication) that explain parts of their phenotypes other than elevated MMA. ConclusionGenome and RNA sequencing did not detect any additional putative causal genetic defects in known cobalamin genes following somatic cell studies and the use of a targeted NGS panel. They did detect pathogenic variants in other genes in three patients that explained some aspects of their clinical presentation.

IPSC Models of Chromosome 15Q Imprinting Disorders: From Disease Modeling to Therapeutic Strategies.

The chromosome 15q11-q13 region of the human genome is regulated by genomic imprinting, an epigenetic phenomenon in which genes are expressed exclusively from one parental allele. Several genes within the 15q11-q13 region are expressed exclusively from the paternally inherited chromosome 15. At least one gene UBE3A, shows exclusive expression of the maternal allele, but this allele-specific expression is restricted to neurons. The appropriate regulation of imprinted gene expression across chromosome 15q11-q13 has important implications for human disease. Three different neurodevelopmental disorders result from aberrant expression of imprinted genes in this region: Prader-Willi syndrome (PWS), Angelman syndrome (AS), and 15q duplication syndrome.

Change in serotonergic modulation contributes to the synaptic imbalance of neuronal circuit at the prefrontal cortex in the 15q11-13 duplication mouse model of autism

The prefrontal cortex (PFC) has been extensively studied in autism spectrum disorder (ASD) in an attempt to understand the deficits in executive and other higher brain functions related to sociability and emotion. Disruption of the excitatory/inhibitory (E/I) balance of cortical circuits is thought to underlie the pathophysiology of ASD. Recently, we showed that 15q dup mice (a model for ASD with human chromosome 15q11-13 paternal duplication) exhibit disruption of the E/I balance in layer 2/3 pyramidal neurons of the somatosensory cortex due to a decrease in the number of inhibitory synapses. However, whether there is a pathological abnormality in E/I balance in the PFC of 15q dup mice remains unknown. In this study, we found that 15q dup facilitates the activity-induced LTP of glutamate synapses onto layer 5 pyramidal neurons by shifting the E/I balance to an excitatory state, which this was associated with differences in synaptic glutamatergic and GABAergic inputs onto GABAergic fast-spiking interneurons (FSINs). Furthermore, we found that FSIN excitability was well-modulated and regulated by the constitutive activation of 5-HT2 receptors in PFC microcircuits. These results provide new insights into the cellular mechanisms underlying maintenance of optimal E/I balance in the PFC.

Altered microbiota composition reflects enhanced communication in 15q11-13 CNV mice

Autism spectrum disorder (ASD) is a complex and heterogeneous neurodevelopmental disorder. In addition to the core symptoms of ASD, many patients with ASD also show comorbid gut dysbiosis, which may lead to various gastrointestinal (GI) problems. Intriguingly, there is evidence that gut microbiota communicate with the central nervous system to modulate behavioral output through the gut-brain axis. To investigate how the microbiota composition is changed in ASD and to identify which microbes are involved in autistic behaviors, we performed a 16S rRNA gene-based metagenomics analysis in an ASD mouse model. Here, we focused on a model with human 15q11-13 duplication (15q dup), the most frequent chromosomal aberration or copy number variation found in ASD. Species diversity of the microbiome was significantly decreased in 15q dup mice. A combination of antibiotics treatment and behavioral analysis showed that neomycin improved social communication in 15q dup mice. Furthermore, comparison of the microbiota composition of mice treated with different antibiotics enabled us to identify beneficial operational taxonomic units (OTUs) for ultrasonic vocalization.

Possible Phenotypic Consequences of Structural Differences in Idic(15) in a Small Cohort of Patients.

Among human supernumerary marker chromosomes, the occurrence of isodicentric form of 15 origin is relatively well known due to its high frequency, both in terms of gene content and associated clinical symptoms. The associated epilepsy and autism are typically more severe than in cases with interstitial 15q duplication, despite copy number gain of approximately the same genomic region. Other mechanisms besides segmental aneuploidy and epigenetic changes may also cause this difference. Among the factors influencing the expression of members of the GABAA gene cluster, the imprinting effect and copy number differences has been debated. Limited numbers of studies investigate factors influencing the interaction of GABAA cluster homologues. Five isodicentric (15) patients are reported with heterogeneous symptoms, and structural differences of their isodicentric chromosomes based on array comparative genomic hybridization results. Relations between the structure and the heterogeneous clinical picture are discussed, raising the possibility that the structure of the isodicentric (15), which has an asymmetric breakpoint and consequently a lower copy number segment, would be the basis of the imbalance of the GABAA homologues. Studies of trans interaction and regulation of GABAA cluster homologues are needed to resolve this issue, considering copy number differences within the isodicentric chromosome 15.

Mosaic embryo diagnosis correlated with abnormal 15q duplication syndrome in offspring

Mosaic embryo diagnosis correlated with abnormal 15q duplication syndrome in offspring

Mechanisms underlying the EEG biomarker in Dup15q syndrome

BackgroundDuplications of 15q11.2-q13.1 (Dup15q syndrome), including the paternally imprinted gene UBE3A and three nonimprinted gamma-aminobutyric acid type-A (GABAA) receptor genes, are highly penetrant for neurodevelopmental disorders such as autism spectrum disorder (ASD). To guide targeted treatments of Dup15q syndrome and other forms of ASD, biomarkers are needed that reflect molecular mechanisms of pathology. We recently described a beta EEG phenotype of Dup15q syndrome, but it remains unknown which specific genes drive this phenotype.MethodsTo test the hypothesis that UBE3A overexpression is not necessary for the beta EEG phenotype, we compared EEG from a reference cohort of children with Dup15q syndrome (n = 27) to (1) the pharmacological effects of the GABAA modulator midazolam (n = 12) on EEG from healthy adults, (2) EEG from typically developing (TD) children (n = 14), and (3) EEG from two children with duplications of paternal 15q (i.e., the UBE3A-silenced allele).ResultsPeak beta power was significantly increased in the reference cohort relative to TD controls. Midazolam administration recapitulated the beta EEG phenotype in healthy adults with a similar peak frequency in central channels (f = 23.0 Hz) as Dup15q syndrome (f = 23.1 Hz). Both paternal Dup15q syndrome cases displayed beta power comparable to the reference cohort.ConclusionsOur results suggest a critical role for GABAergic transmission in the Dup15q syndrome beta EEG phenotype, which cannot be explained by UBE3A dysfunction alone. If this mechanism is confirmed, the phenotype may be used as a marker of GABAergic pathology in clinical trials for Dup15q syndrome.

Concurrent SHORT syndrome and 3q duplication syndrome

Concurrent SHORT syndrome and 3q duplication syndrome

Prenatal identification of partial 3q duplication syndrome

BackgroundThe 3q duplication syndrome is a result of duplication of a large fragment of the long arm of chromosome 3, mainly 3q21-qter, and in most cases it is diagnosed only after birth. The phenotypic consequences resulting from genetic imbalance are an important source of information for genetic counselling, especially in prenatal diagnostics. However, in most cases it is impossible to define them precisely because the final clinical presentation is a result of an overlap, usually due to different sizes of deletions and/or duplications not only chromosome 3 but also of translocation partner chromosome. In this article, we present a prenatal diagnosis of the 3q duplication syndrome in a foetus, arising from a balanced insertion ins (7,3)(q21.2;q12.3q29) carried by the mother.Case presentationThe article presents a case of a 29-year-old woman referred to the Genetic Outpatient Clinic for consultation in the 12th week of her fifth pregnancy with a diagnosis of generalised hydrops foetalis. The analysis of karyotype using GTG technique and FISH allowed diagnosis of a balanced aberration in the mother, and determined the type of chromosomal rearrangement, which allowed the identification of the origin of the additional genetic material in the foetus and the previous malformed child of the same couple. The use of molecular karyotyping techniques (FISH and aCGH) allowed a precise determination of the size of the imbalanced fragments in the affected siblings.ConclusionsThe aCGH technique is particularly valuable for the diagnostics of submicroscopic deletions and duplications, if no imbalanced chromosomal aberrations are detected by routine cytogenetic tests. It is also a valuable technique for identifying and fully characterizing genetic material of unknown origin, which can’t be identified using routine cytogenetic techniqes. However, it does not allow identification of balanced aberrations in carriers.

UBE3A-mediated PTPA ubiquitination and degradation regulate PP2A activity and dendritic spine morphology

Deficiency in the E3 ubiquitin ligase UBE3A leads to the neurodevelopmental disorder Angelman syndrome (AS), while additional dosage of UBE3A is linked to autism spectrum disorder. The mechanisms underlying the downstream effects of UBE3A gain or loss of function in these neurodevelopmental disorders are still not well understood, and effective treatments are lacking. Here, using stable-isotope labeling of amino acids in mammals and ubiquitination assays, we identify PTPA, an activator of protein phosphatase 2A (PP2A), as a bona fide ubiquitin ligase substrate of UBE3A. Maternal loss of Ube3a (Ube3am-/p+) increased PTPA level, promoted PP2A holoenzyme assembly, and elevated PP2A activity, while maternal 15q11-13 duplication containing Ube3a down-regulated PTPA level and lowered PP2A activity. Reducing PTPA level in vivo restored the defects in dendritic spine maturation in Ube3am-/p+ mice. Moreover, pharmacological inhibition of PP2A activity with the small molecule LB-100 alleviated both reduction in excitatory synaptic transmission and motor impairment in Ube3am-/p+ mice. Together, our results implicate a critical role of UBE3A-PTPA-PP2A signaling in the pathogenesis of UBE3A-related disorders and suggest that PP2A-based drugs could be potential therapeutic candidates for treatment of UBE3A-related disorders.

Spurious elevations of total vitamin B12 identified with polyethylene glycol precipitation procedure

Our laboratory has classified patients with methylmalonic aciduria using somatic cell studies for over four decades. We have accumulated 127 fibroblast lines from patients with persistent elevated methylmalonic acid (MMA) levels in which no genetic cause could be identified. Cultured fibroblasts from 26 of these patients had low [14C]propionate incorporation into macromolecules, possibly reflecting decreased methylmalonyl-CoA mutase function.Genome sequencing (GS), copy-number variation (CNV) analysis, and RNA sequencing were performed on genomic DNA and complementary DNA (cDNA) from these 26 patients.No patient had two pathogenic variants in any gene associated with cobalamin metabolism. Nine patients had heterozygous variants of unknown significance previously identified by a next-generation sequencing (NGS) panel targeting cobalamin metabolic genes. Three patients had pathogenic changes in genes not associated with cobalamin metabolism (PCCA, EPCAM, and a 17q12 duplication) that explain parts of their phenotypes other than elevated MMA.Genome and RNA sequencing did not detect any additional putative causal genetic defects in known cobalamin genes following somatic cell studies and the use of a targeted NGS panel. They did detect pathogenic variants in other genes in three patients that explained some aspects of their clinical presentation.

A genome-wide enhancer/suppressor screen for Dube3a interacting genes in Drosophila melanogaster

The genetics underlying autism spectrum disorder (ASD) are complex. Approximately 3–5% of ASD cases arise from maternally inherited duplications of 15q11.2-q13.1, termed Duplication 15q syndrome (Dup15q). 15q11.2-q13.1 includes the gene UBE3A which is believed to underlie ASD observed in Dup15q syndrome. UBE3A is an E3 ubiquitin ligase that targets proteins for degradation and trafficking, so finding UBE3A substrates and interacting partners is critical to understanding Dup15q ASD. In this study, we take an unbiased genetics approach to identify genes that genetically interact with Dube3a, the Drosophila melanogaster homolog of UBE3A. We conducted an enhancer/suppressor screen using a rough eye phenotype produced by Dube3a overexpression with GMR-GAL4. Using the DrosDel deficiency kit, we identified 3 out of 346 deficiency lines that enhanced rough eyes when crossed to two separate Dube3a overexpression lines, and subsequently identified IA2, GABA-B-R3, and lola as single genes responsible for rough eye enhancement. Using the FlyLight GAL4 lines to express uas-Dube3a + uas-GFP in the endogenous lola pattern, we observed an increase in the GFP signal compared to uas-GFP alone, suggesting a transcriptional co-activation effect of Dube3a on the lola promoter region. These findings extend the role of Dube3a/UBE3A as a transcriptional co-activator, and reveal new Dube3a interacting genes.

22q11.2 duplications in a UK cohort with bladder exstrophy-epispadias complex.

The bladder exstrophy-epispadias complex (BEEC) comprises of a spectrum of anterior midline defects, all affecting the lower urinary tract, the external genitalia, and the bony pelvis. In extreme cases, the gastrointestinal tract is also affected. The pathogenesis of BEEC is unclear but chromosomal aberrations have been reported. In particular, duplications of 22q11.2 have been identified in eight unrelated individuals with BEEC. The current study aimed to identify chromosomal copy number variants in BEEC. Analyses was performed using the Affymetrix Genome-wide SNP6.0 assay in 92 unrelated patients cared for by two UK pediatric urology centers. Three individuals had a 22q11.2 duplication, a significantly higher number than that found in a control group of 12,500 individuals with developmental delay who had undergone microarray testing (p < .0001). Sequencing of CRKL, implicated in renal tract malformations in DiGeorge syndrome critical region at 22q11, in 89 individuals with BEEC lacking 22q11 duplications revealed no pathogenic variants. To date, 22q11.2 duplication is the genetic variant most commonly associated with BEEC. This is consistent with the hypothesis that altered expression of a single, yet to be defined, gene therein is critical to the pathogenesis of this potentially devastating congenital disorder.

M52 - CONVERGENT METHYLOMIC SIGNATURES BETWEEN AUTISM ASSOCIATED WITH DUPLICATIONS OF CHROMOSOME 15Q AND IDIOPATHIC AUTISM

Background Autism Spectrum Disorder (ASD) includes a spectrum of genetically and clinically complex neurodevelopmental disorders. Findings from recent discordant monozygotic twins and post-mortem brain studies suggest that altered epigenetic processes, including DNA methylation and histone acetylation, are involved in the etiology of ASD. This study presents, to our knowledge, the largest post-mortem genome-wide DNA methylation analyses of autism patients, including idiopathic ASD and chromosome 15q11.2-13.1 duplication syndrome (dup15q) carriers, and matched controls. Methods We performed methylomic profiling in human post-mortem brain tissues using samples from three brain regions (dorsolateral prefrontal cortex, primary auditory cortex and cerebellum) of idiopathic autism cases, dup15q cariers and matched controls using the Illumina Infinium HumanMethylation450 array. Following stringent quality control and data pre-processing ASD-associated differential methylation analyses were performed at both probe-wise and region-wise levels. Results Our analyses revealed ASD-associated dysregulation of DNA methylation at multiple loci, with a large number of cross-cortex significant differentially methylated probes. In addition, although the differential methylation observed in dup15q samples were much more pronounced than those from iASD samples, we observed a convergent of methylomic signatures between autism associated with duplications of chromosome 15q and idiopathic autism. Discussion This epigenome-wide study of autism using post-mortem tissues represents the most comprehensive study to date. Findings from this study further support a role of altered epigenetics signatures in ASD and provide novel insight that idiopathic ASD and a genetically defined form of ASD (i.e. dup15q) carry similar underlying epigenetics changes.

Fetal neural stem cells from a mouse model of 15q11-13 duplication syndrome exhibit altered differentiation into neurons and astrocytes

The duplication of human chromosome 15q11-13 is known to be associated with an estimated 1.1% of autism cases. Here, we investigated whether differentiation into neurons and astrocytes is altered in fetal neural stem cells (FNSCs) isolated from the mouse model of 15q11-13 duplication syndrome (patDp/+ mice). In patDp/+ mice-derived FNSCs, multipotency was maintained for a longer period, the population of neurons was downregulated, and that of astrocytes was upregulated significantly after differentiation induction. These results suggest that the dysregulation of FNSCs differentiation could affect cortical development and behavioral deficits in the early postnatal stage shown in the patDp/+ mice.

Phase II Trial of Combination of Elotuzumab, Lenalidomide, and Dexamethasone in High-Risk Smoldering Multiple Myeloma

Phase II Trial of Combination of Elotuzumab, Lenalidomide, and Dexamethasone in High-Risk Smoldering Multiple Myeloma

Cytogenomic characterization of 1q43q44 deletion associated with 4q32.1q35.2 duplication and phenotype correlation

BackgroundMicrodeletion of 1q43q44 causes a syndrome characterized by intellectual disability (ID), speech delay, seizures, microcephaly (MIC), corpus callosum abnormalities (CCA) and characteristic facial features. Duplication of 4q is presented with minor to severe ID, MIC and facial dysmorphism. We aimed to verify the correlation between genotype/phenotype in a patient with 1q43q44 deletion associated with 4q32.1q35.2 duplication.Case presentationWe report on a 3 year-old female patient with delayed motor and mental milestones, MIC and facial dysmorphism. She is a child of non-consanguineous parents and no similarly affected family members. CT brain showed abnormal gyral patterns, hypogenesis of corpus callosum and bilateral deep Sylvian fissure. Electroencephalogram showed frontotemporal epileptogenic focus. Her karyotype was revealed as 46,XX,add(1)(q44). Fluorescence in situ hybridization (FISH) using whole chromosome paint (WCP1) and subtelomere 1q revealed that the add segment was not derived from chromosome 1 and there was the deletion of subtelomere 1q. Multiple ligation probe amplification (MLPA) subtelomere kit revealed the deletion of 1q and duplication of 4q. Array CGH demonstrated the 6.5 Mb deletion of 1q and 31 Mb duplication of chromosome 4q.ConclusionThe phenotype of our patient mainly reflects the effects of haploinsufficiency of AKT3, HNRNPU, ZBTB18 genes associated with duplication of GLRA3, GMP6A, HAND2 genes. Patients presented with ID, seizures, MIC together with CCA are candidates for prediction of 1q43q44 microdeletion and cytogenomic analysis.