A bipartite boundary element restrictsUBE3Aimprinting to mature neurons

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of function from the maternal allele ofUBE3A, a gene encoding an E3 ubiquitin ligase.UBE3Ais only expressed from the maternally inherited allele in mature human neurons due to tissue-specific genomic imprinting. Imprinted expression ofUBE3Ais restricted to neurons by expression ofUBE3A antisense transcript(UBE3A-ATS) from the paternally inherited allele, which silences the paternal allele ofUBE3Aincis. However, the mechanism restrictingUBE3A-ATSexpression andUBE3Aimprinting to neurons is not understood. We used CRISPR/Cas9-mediated genome editing to functionally define a bipartite boundary element critical for neuron-specific expression ofUBE3A-ATSin humans. Removal of this element led to up-regulation ofUBE3A-ATSwithout repressing paternalUBE3A. However, increasing expression ofUBE3A-ATSin the absence of the boundary element resulted in full repression of paternalUBE3A, demonstrating thatUBE3Aimprinting requires both the loss of function from the boundary element as well as the up-regulation ofUBE3A-ATS. These results suggest that manipulation of the competition betweenUBE3A-ATSandUBE3Amay provide a potential therapeutic approach for AS.

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BIOLOGICAL SCIENCES: Genetics

10.1101/404061 ◽

2018 ◽

Author(s):

Jack S. Hsiao ◽

Noelle D. Germain ◽

Andrea Wilderman ◽

Christopher Stoddard ◽

Luke A. Wojenski ◽

...

Keyword(s):

Boundary Element ◽

Angelman Syndrome ◽

Antisense Transcript ◽

Neurodevelopmental Disorder ◽

Boundary Function ◽

Paternal Allele ◽

Loss Of Function ◽

Maternal Allele ◽

Specific Expression ◽

Human Neurons

ABSTRACTAngelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of function from the maternal allele of UBE3A, a gene encoding an E3 ubiquitin ligase. UBE3A is only expressed from the maternally-inherited allele in mature human neurons due to tissue-specific genomic imprinting. Imprinted expression of UBE3A is restricted to neurons by expression of UBE3A antisense transcript (UBE3A-ATS) from the paternally-inherited allele, which silences the paternal allele of UBE3A in cis. However, the mechanism restricting UBE3A-ATS expression and UBE3A imprinting to neurons is not understood. We used CRISPR/Cas9-mediated genome editing to functionally define a bipartite boundary element critical for neuron-specific expression of UBE3A-ATS in humans. Removal of this element led to upregulation of UBE3A-ATS without repressing paternal UBE3A. However, increasing expression of UBE3A-ATS in the absence of the boundary element resulted in full repression of paternal UBE3A, demonstrating that UBE3A imprinting requires both the loss of function from the boundary element as well as upregulation of UBE3A-ATS. These results suggest that manipulation of the competition between UBE3A-ATS and UBE3A may provide a potential therapeutic approach for AS.SIGNIFICANCE STATEMENTAngelman syndrome is a neurodevelopmental disorder caused by loss of function from the maternal allele of UBE3A, an imprinted gene. The paternal allele of UBE3A is silenced by a long, non-coding antisense transcript in mature neurons. We have identified a boundary element that stops the transcription of the antisense transcript in human pluripotent stem cells, and thus restricts UBE3A imprinted expression to neurons. We further determined that UBE3A imprinting requires both the loss of the boundary function and sufficient expression of the antisense transcript to silence paternal UBE3A. These findings provide essential details about the mechanisms of UBE3A imprinting that may suggest additional therapeutic approaches for Angelman syndrome.

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Multiple Genomic Events Altering Hominin SIGLEC Biology and Innate Immunity Predated the Common Ancestor of Humans and Archaic Hominins

Genome Biology and Evolution ◽

10.1093/gbe/evaa125 ◽

2020 ◽

Vol 12 (7) ◽

pp. 1040-1050

Author(s):

Naazneen Khan ◽

Marc de Manuel ◽

Stephane Peyregne ◽

Raymond Do ◽

Kay Prufer ◽

...

Keyword(s):

Innate Immunity ◽

Common Ancestor ◽

Loss Of Function ◽

Gene Encoding ◽

Specific Expression ◽

Functional Changes ◽

Almost All ◽

Molecular Patterns ◽

Genomic Cluster ◽

Human Specific

Abstract Human-specific pseudogenization of the CMAH gene eliminated the mammalian sialic acid (Sia) Neu5Gc (generating an excess of its precursor Neu5Ac), thus changing ubiquitous cell surface “self-associated molecular patterns” that modulate innate immunity via engagement of CD33-related-Siglec receptors. The Alu-fusion-mediated loss-of-function of CMAH fixed ∼2–3 Ma, possibly contributing to the origins of the genus Homo. The mutation likely altered human self-associated molecular patterns, triggering multiple events, including emergence of human-adapted pathogens with strong preference for Neu5Ac recognition and/or presenting Neu5Ac-containing molecular mimics of human glycans, which can suppress immune responses via CD33-related-Siglec engagement. Human-specific alterations reported in some gene-encoding Sia-sensing proteins suggested a “hotspot” in hominin evolution. The availability of more hominid genomes including those of two extinct hominins now allows full reanalysis and evolutionary timing. Functional changes occur in 8/13 members of the human genomic cluster encoding CD33-related Siglecs, all predating the human common ancestor. Comparisons with great ape genomes indicate that these changes are unique to hominins. We found no evidence for strong selection after the Human–Neanderthal/Denisovan common ancestor, and these extinct hominin genomes include almost all major changes found in humans, indicating that these changes in hominin sialobiology predate the Neanderthal–human divergence ∼0.6 Ma. Multiple changes in this genomic cluster may also explain human-specific expression of CD33rSiglecs in unexpected locations such as amnion, placental trophoblast, pancreatic islets, ovarian fibroblasts, microglia, Natural Killer(NK) cells, and epithelia. Taken together, our data suggest that innate immune interactions with pathogens markedly altered hominin Siglec biology between 0.6 and 2 Ma, potentially affecting human evolution.

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Immune Dysfunction in Rett Syndrome Patients Revealed by High Levels of Serum Anti-N(Glc) IgM Antibody Fraction

Journal of Immunology Research ◽

10.1155/2014/260973 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6 ◽

Cited By ~ 10

Author(s):

Anna Maria Papini ◽

Francesca Nuti ◽

Feliciana Real-Fernandez ◽

Giada Rossi ◽

Caterina Tiberi ◽

...

Keyword(s):

Immune System ◽

Rett Syndrome ◽

Pervasive Developmental Disorders ◽

Developmental Disorders ◽

Neurodevelopmental Disorder ◽

Loss Of Function ◽

Gene Encoding ◽

Antibody Recognition ◽

Forkhead Box ◽

Significant Difference

Rett syndrome (RTT), a neurodevelopmental disorder affecting exclusively (99%) female infants, is associated with loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2) and, more rarely, cyclin-dependent kinase-like 5 (CDKL5) and forkhead box protein G1 (FOXG1). In this study, we aimed to evaluate the function of the immune system by measuring serum immunoglobulins (IgG and IgM) in RTT patients (n=53) and, by comparison, in age-matched children affected by non-RTT pervasive developmental disorders (non-RTT PDD) (n=82) and healthy age-matched controls (n=29). To determine immunoglobulins we used both a conventional agglutination assay and a novel ELISA based on antibody recognition by a surrogate antigen probe, CSF114(Glc), a syntheticN-glucosylated peptide. Both assays provided evidence for an increase in IgM titer, but not in IgG, in RTT patients relative to both healthy controls and non-RTT PDD patients. The significant difference in IgM titers between RTT patients and healthy subjects in the CSF114(Glc) assay (P=0.001) suggests that this procedure specifically detects a fraction of IgM antibodies likely to be relevant for the RTT disease. These findings offer a new insight into the mechanism underlying the Rett disease as they unveil the possible involvement of the immune system in this pathology.

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Subclinical Inflammatory Status in Rett Syndrome

Mediators of Inflammation ◽

10.1155/2014/480980 ◽

2014 ◽

Vol 2014 ◽

pp. 1-13 ◽

Cited By ~ 40

Author(s):

Alessio Cortelazzo ◽

Claudio De Felice ◽

Roberto Guerranti ◽

Cinzia Signorini ◽

Silvia Leoncini ◽

...

Keyword(s):

Gene Mutation ◽

Rett Syndrome ◽

De Novo ◽

Clinical Chemistry ◽

Neurodevelopmental Disorder ◽

Loss Of Function ◽

Gene Encoding ◽

Protein Levels ◽

Large Deletions ◽

Inflammatory Status

Inflammation has been advocated as a possible common central mechanism for developmental cognitive impairment. Rett syndrome (RTT) is a devastating neurodevelopmental disorder, mainly caused byde novoloss-of-function mutations in the gene encoding MeCP2. Here, we investigated plasma acute phase response (APR) in stage II (i.e., “pseudo-autistic”) RTT patients by routine haematology/clinical chemistry and proteomic 2-DE/MALDI-TOF analyses as a function of four majorMECP2gene mutation types (R306C, T158M, R168X, and large deletions). Elevated erythrocyte sedimentation rate values (median 33.0 mm/h versus 8.0 mm/h,P<0.0001) were detectable in RTT, whereas C-reactive protein levels were unchanged (P=0.63). The 2-DE analysis identified significant changes for a total of 17 proteins, the majority of which were categorized as APR proteins, either positive (n=6spots) or negative (n=9spots), and to a lesser extent as proteins involved in the immune system (n=2spots), with some proteins having overlapping functions on metabolism (n=7spots). The number of protein changes was proportional to the severity of the mutation. Our findings reveal for the first time the presence of a subclinical chronic inflammatory status related to the “pseudo-autistic” phase of RTT, which is related to the severity carried by theMECP2gene mutation.

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Antisense oligonucleotides targeting UBE3A-ATS restore expression of UBE3A by relieving transcriptional interference

10.1101/2021.07.09.451826 ◽

2021 ◽

Author(s):

Noelle D. Germain ◽

Dea Gorka ◽

Ryan Drennan ◽

Amanda Whipple ◽

Paymaan Jafar-nejad ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Antisense Oligonucleotides ◽

Noncoding Rna ◽

Neurodevelopmental Disorder ◽

Paternal Allele ◽

Transcriptional Interference ◽

Loss Of Function ◽

Definitive Evidence ◽

Promising Therapeutic Approach ◽

In Cis

Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by loss of function of the maternally inherited UBE3A allele. In neurons, the paternal allele of UBE3A is silenced in cis by the long noncoding RNA, UBE3A-ATS. Unsilencing paternal UBE3A by reducing UBE3A-ATS is a promising therapeutic approach for the treatment of AS. Here we show that targeted cleavage of UBE3A-ATS using antisense oligonucleotides (ASOs) restores UBE3A and rescues electrophysiological phenotypes in human AS neurons. We demonstrate that cleavage of UBE3A-ATS results in termination of its transcription by displacement of RNA Polymerase II. Reduced transcription of UBE3A-ATS allows transcription of UBE3A to proceed to completion, providing definitive evidence for the transcriptional interference model of paternal UBE3A silencing. These insights into the mechanism by which ASOs restore UBE3A inform the future development of nucleotide-based approaches for the treatment of AS, including alternative strategies for cleaving UBE3A-ATS that can be developed for long-term restoration of UBE3A function.

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Abstract 198: Aberrant Endothelial-myocardial Crosstalk Causes Hypertrophy in Noonan Syndrome With Multiple Lentigines

Circulation Research ◽

10.1161/res.117.suppl_1.198 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Jessica Lauriol ◽

Janel R Cabrera ◽

Gabriel C Segarra ◽

Meaghan E Flessa ◽

Lauren E Miller ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Noonan Syndrome ◽

Cardiac Development ◽

Tyrosine Phosphatase ◽

Adult Onset ◽

Loss Of Function ◽

Gene Encoding ◽

Specific Expression ◽

Underlying Mechanisms ◽

Multiple Lentigines

Congenital heart disease (CHD) is the most common birth defect worldwide; however, underlying mechanisms remain unknown. Loss-of-function mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP2, are implicated in CHD and cause Noonan Syndrome with Multiple Lentigines (NSML). NSML presents with multiple cardiac defects, including hypertrophy. Here, we found that the NSML-associated adult-onset cardiac hypertrophy stems from aberrant signaling originating from developing endocardium. Embryonic NSML hearts showed diminished trabeculation and valvular hyperplasia, defects recapitulated in endocardial-, but not myocardial- or neural crest-, specific NSML mice. NSML hearts also developed ventricular septal defects, a phenotype reproduced only in myocardial-specific NSML hearts, suggesting NSML mutations have both cell autonomous and non-autonomous functions in cardiac development. Importantly, endocardial-specific expression of NSML was sufficient to induce adult-onset cardiac hypertrophy. Mechanistically, we observed aberrant AKT activity in NSML embryos, with decreased downstream FOXP1/FGF and NOTCH1/EPHB2 signaling, two pathways necessary for reciprocal crosstalk between developing endocardium and myocardium. Taken together, our data provide the first functional and disease-based evidence to suggest that critical mechanisms exist to control endocardial-myocardial crosstalk, the aberrant regulation of which may lead to CHD and adult-onset cardiac disease.

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Human SYNGAP1 Regulates the Development of Neuronal Activity by Controlling Dendritic and Synaptic Maturation

10.1101/2020.06.01.127613 ◽

2020 ◽

Author(s):

Nerea Llamosas ◽

Vineet Arora ◽

Ridhima Vij ◽

Murat Kilinc ◽

Lukasz Bijoch ◽

...

Keyword(s):

Protein Expression ◽

Neurodevelopmental Disorder ◽

Autism Spectrum ◽

Synaptic Activity ◽

Global Developmental Delay ◽

Excitatory Synapses ◽

Loss Of Function ◽

Biological Studies ◽

Human Neurons ◽

And Function

AbstractSYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function variants driving formation of larger dendritic spines and stronger glutamatergic transmission. However, studies to date have been limited to mouse and rat neurons. Therefore, it remains unknown how SYNGAP1 loss-of-function impacts the development and function of human neurons. To address this, we employed CRISPR/Cas9 technology to ablate SYNGAP1 protein expression in neurons derived from a human induced pluripotent stem cell line (hiPSC). Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger neurons compared to those derived from isogenic controls. Consistent with larger dendritic fields, we also observed a greater number of morphologically defined excitatory synapses in cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, distributed network spiking activity appeared earlier, was substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from hiPSCs, which influences how activity develops within nascent neural networks. Alterations to this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-related disorders.

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Gain-of-function GABRB3 variants identified in vigabatrin-hypersensitive epileptic encephalopathies

Brain Communications ◽

10.1093/braincomms/fcaa162 ◽

2020 ◽

Vol 2 (2) ◽

Author(s):

Nathan L Absalom ◽

Vivian W Y Liao ◽

Kavitha Kothur ◽

Dinesh C Indurthi ◽

Bruce Bennetts ◽

...

Keyword(s):

De Novo ◽

Drug Effects ◽

Receptor Expression ◽

Aminobutyric Acid ◽

Loss Of Function ◽

Molecular Phenotype ◽

Gain Of Function ◽

Gene Encoding ◽

Epileptic Encephalopathies ◽

Receptor Phenotype

Abstract Variants in the GABRB3 gene encoding the β3-subunit of the γ-aminobutyric acid type A ( receptor are associated with various developmental and epileptic encephalopathies. Typically, these variants cause a loss-of-function molecular phenotype whereby γ-aminobutyric acid has reduced inhibitory effectiveness leading to seizures. Drugs that potentiate inhibitory GABAergic activity, such as nitrazepam, phenobarbital or vigabatrin, are expected to compensate for this and thereby reduce seizure frequency. However, vigabatrin, a drug that inhibits γ-aminobutyric acid transaminase to increase tonic γ-aminobutyric acid currents, has mixed success in treating seizures in patients with GABRB3 variants: some patients experience seizure cessation, but there is hypersensitivity in some patients associated with hypotonia, sedation and respiratory suppression. A GABRB3 variant that responds well to vigabatrin involves a truncation variant (p.Arg194*) resulting in a clear loss-of-function. We hypothesized that patients with a hypersensitive response to vigabatrin may exhibit a different γ-aminobutyric acid A receptor phenotype. To test this hypothesis, we evaluated the phenotype of de novo variants in GABRB3 (p.Glu77Lys and p.Thr287Ile) associated with patients who are clinically hypersensitive to vigabatrin. We introduced the GABRB3 p.Glu77Lys and p.Thr287Ile variants into a concatenated synaptic and extrasynaptic γ-aminobutyric acid A receptor construct, to resemble the γ-aminobutyric acid A receptor expression by a patient heterozygous for the GABRB3 variant. The mRNA of these constructs was injected into Xenopus oocytes and activation properties of each receptor measured by two-electrode voltage clamp electrophysiology. Results showed an atypical gain-of-function molecular phenotype in the GABRB3 p.Glu77Lys and p.Thr287Ile variants characterized by increased potency of γ-aminobutyric acid A without change to the estimated maximum open channel probability, deactivation kinetics or absolute currents. Modelling of the activation properties of the receptors indicated that either variant caused increased chloride flux in response to low concentrations of γ-aminobutyric acid that mediate tonic currents. We therefore propose that the hypersensitivity reaction to vigabatrin is a result of GABRB3 variants that exacerbate GABAergic tonic currents and caution is required when prescribing vigabatrin. In contrast, drug strategies increasing tonic currents in loss-of-function variants are likely to be a safe and effective therapy. This study demonstrates that functional genomics can explain beneficial and adverse anti-epileptic drug effects, and propose that vigabatrin should be considered in patients with clear loss-of-function GABRB3 variants.

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Emerging Role of ODC1 in Neurodevelopmental Disorders and Brain Development

Genes ◽

10.3390/genes12040470 ◽

2021 ◽

Vol 12 (4) ◽

pp. 470

Author(s):

Jeremy W. Prokop ◽

Caleb P. Bupp ◽

Austin Frisch ◽

Stephanie M. Bilinovich ◽

Daniel B. Campbell ◽

...

Keyword(s):

Brain Development ◽

Neural Progenitor Cells ◽

Neurodevelopmental Disorder ◽

Fetal Brain ◽

Missense Variant ◽

Neural Progenitor ◽

Loss Of Function ◽

Gain Of Function ◽

Systematic Analysis ◽

Function Expression

Ornithine decarboxylase 1 (ODC1 gene) has been linked through gain-of-function variants to a rare disease featuring developmental delay, alopecia, macrocephaly, and structural brain anomalies. ODC1 has been linked to additional diseases like cancer, with growing evidence for neurological contributions to schizophrenia, mood disorders, anxiety, epilepsy, learning, and suicidal behavior. The evidence of ODC1 connection to neural disorders highlights the need for a systematic analysis of ODC1 genotype-to-phenotype associations. An analysis of variants from ClinVar, Geno2MP, TOPMed, gnomAD, and COSMIC revealed an intellectual disability and seizure connected loss-of-function variant, ODC G84R (rs138359527, NC_000002.12:g.10444500C > T). The missense variant is found in ~1% of South Asian individuals and results in 2.5-fold decrease in enzyme function. Expression quantitative trait loci (eQTLs) reveal multiple functionally annotated, non-coding variants regulating ODC1 that associate with psychiatric/neurological phenotypes. Further dissection of RNA-Seq during fetal brain development and within cerebral organoids showed an association of ODC1 expression with cell proliferation of neural progenitor cells, suggesting gain-of-function variants with neural over-proliferation and loss-of-function variants with neural depletion. The linkage from the expression data of ODC1 in early neural progenitor proliferation to phenotypes of neurodevelopmental delay and to the connection of polyamine metabolites in brain function establish ODC1 as a bona fide neurodevelopmental disorder gene.

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