Human Haploinsufficiency Research Articles

ARID1B maintains mesenchymal stem cell quiescence via inhibition of BCL11B-mediated non-canonical Activin signaling

ARID1B haploinsufficiency in humans causes Coffin-Siris syndrome, associated with developmental delay, facial dysmorphism, and intellectual disability. The role of ARID1B has been widely studied in neuronal development, but whether it also regulates stem cells remains unknown. Here, we employ scRNA-seq and scATAC-seq to dissect the regulatory functions and mechanisms of ARID1B within mesenchymal stem cells (MSCs) using the mouse incisor model. We reveal that loss of Arid1b in the GLI1+ MSC lineage disturbs MSCs’ quiescence and leads to their proliferation due to the ectopic activation of non-canonical Activin signaling via p-ERK. Furthermore, loss of Arid1b upregulates Bcl11b, which encodes a BAF complex subunit that modulates non-canonical Activin signaling by directly regulating the expression of activin A subunit, Inhba. Reduction of Bcl11b or non-canonical Activin signaling restores the MSC population in Arid1b mutant mice. Notably, we have identified that ARID1B suppresses Bcl11b expression via specific binding to its third intron, unveiling the direct inter-regulatory interactions among BAF subunits in MSCs. Our results demonstrate the vital role of ARID1B as an epigenetic modifier in maintaining MSC homeostasis and reveal its intricate mechanistic regulatory network in vivo, providing novel insights into the linkage between chromatin remodeling and stem cell fate determination.

MYRF: A unique transmembrane transcription factor- from proteolytic self-processing to its multifaceted roles in animal development.

The Myelin Regulator Factor (MYRF) is a master regulator governing myelin formation and maintenance in the central nervous system. The conservation of MYRF across metazoans and its broad tissue expression suggest it has functions extending beyond the well-established role in myelination. Loss of MYRF results in developmental lethality in both invertebrates and vertebrates, and MYRF haploinsufficiency in humans causes MYRF-related Cardiac Urogenital Syndrome, underscoring its importance in animal development; however, these mechanisms are largely unexplored. MYRF, an unconventional transcription factor, begins embedded in the membrane and undergoes intramolecular chaperone mediated trimerization, which triggers self-cleavage, allowing its N-terminal segment with an Ig-fold DNA-binding domain to enter the nucleus for transcriptional regulation. Recent research suggests developmental regulation of cleavage, yet the mechanisms remain enigmatic. While some parts of MYRF's structure have been elucidated, others remain obscure, leaving questions about how these motifs are linked to its intricate processing and function.

Comparative analyses of the Smith-Magenis syndrome protein RAI1 in mice and common marmoset monkeys.

Retinoic acid-induced 1 (RAI1) encodes a transcriptional regulator critical for brain development and function. RAI1 haploinsufficiency in humans causes a syndromic autism spectrum disorder known as Smith-Magenis syndrome (SMS). The neuroanatomical distribution of RAI1 has not been quantitatively analyzed during the development of the prefrontal cortex, a brain region critical for cognitive function and social behaviors and commonly implicated in autism spectrum disorders, including SMS. Here, we performed comparative analyses to uncover the evolutionarily convergent and divergent expression profiles of RAI1 in major cell types during prefrontal cortex maturation in common marmoset monkeys (Callithrix jacchus) and mice (Mus musculus). We found that while RAI1 in both species is enriched in neurons, the percentage of excitatory neurons that express RAI1 is higher in newborn mice than in newborn marmosets. By contrast, RAI1 shows similar neural distribution in adult marmosets and adult mice. In marmosets, RAI1 is expressed in several primate-specific cell types, including intralaminar astrocytes and MEIS2-expressing prefrontal GABAergic neurons. At the molecular level, we discovered that RAI1 forms a protein complex with transcription factor 20 (TCF20), PHD finger protein 14 (PHF14), and high mobility group 20A (HMG20A) in the marmoset brain. In vitro assays in human cells revealed that TCF20 regulates RAI1 protein abundance. This work demonstrates that RAI1 expression and protein interactions are largely conserved but with some unique expression in primate-specific cells. The results also suggest that altered RAI1 abundance could contribute to disease features in disorders caused by TCF20 dosage imbalance.

Interneuron-Targeted Disruption of SYNGAP1 Alters Sensory Representations in the Neocortex and Impairs Sensory Learning.

SYNGAP1 haploinsufficiency in humans leads to severe neurodevelopmental disorders characterized by intellectual disability, autism, epilepsy, and sensory processing deficits. However, the circuit mechanisms underlying these disorders are not well understood. In mice, a decrease of SynGAP levels results in cognitive deficits by interfering with the development of excitatory glutamatergic connections. Recent evidence suggests that SynGAP also plays a crucial role in the development and function of GABAergic inhibitory interneurons. Nevertheless, it remains uncertain whether and to what extent the expression of SYNGAP1 in inhibitory interneurons contributes to cortical circuit function and related behaviors. The activity of cortical neurons has not been measured simultaneously with behavior. To address these gaps, we recorded from layer 2/3 neurons in the primary whisker somatosensory cortex (wS1) of mice while they learned to perform a whisker tactile detection task. Our results demonstrate that mice with interneuron-specific SYNGAP1 haploinsufficiency exhibit learning deficits characterized by heightened behavioral responses in the absence of relevant sensory input and premature responses to unrelated sensory stimuli not associated with reward acquisition. These behavioral deficits are accompanied by specific circuit abnormalities within wS1. Interneuron-specific SYNGAP1 haploinsufficiency increases detrimental neuronal correlations directly related to task performance and enhances responses to irrelevant sensory stimuli unrelated to the reward acquisition. In summary, our findings indicate that a reduction of SynGAP in inhibitory interneurons impairs sensory representation in the primary sensory cortex by disrupting neuronal correlations, which likely contributes to the observed cognitive deficits in mice with pan-neuronal SYNGAP1 haploinsufficiency.SIGNIFICANCE STATEMENT SYNGAP1 haploinsufficiency leads to severe neurodevelopmental disorders. The exact nature of neural circuit dysfunction caused by SYNGAP1 haploinsufficiency remains poorly understood. SynGAP plays a critical role in the function of GABAergic inhibitory interneurons as well as glutamatergic pyramidal neurons in the neocortex. Whether and how decreasing SYNGAP1 level in inhibitory interneurons disrupts a behaviorally relevant circuit remains unclear. We measure neural activity and behavior in mice learning a perceptual task. Mice with interneuron-targeted disruption of SYNGAP1 display increased detrimental neuronal correlations and elevated responses to irrelevant sensory inputs, which are related to impaired task performance. These results show that cortical interneuron dysfunction contributes to sensory deficits in SYNGAP1 haploinsufficiency with important implications for identifying therapeutic targets.

Depressed glutamate transporter 1 expression in a mouse model of Dravet syndrome.

Dravet syndrome (DS) is a monogenic, often refractory, epilepsy resultant from SCN1A haploinsufficiency in humans. A novel therapeutic target in DS that can be engaged in isolation or as adjunctive therapy is highly desirable. Here, we demonstrate reduced expression of the rodent glutamate transporter type 1 (GLT-1) in a DS mouse model, and in wild type mouse strains where Scn1a haploinsufficiency is most likely to cause epilepsy, indicating that GLT-1 depression may play a role in DS seizures. As GLT-1 can be upregulated by common and safe FDA-approved medications, this strategy may be an attractive, viable, and novel avenue for DS treatment.

Syngap1 Disruption Induced by Recombination between Inverted loxP Sites Is Associated with Hippocampal Interneuron Dysfunction.

SYNGAP1 haploinsufficiency in humans causes intellectual disability (ID). SYNGAP1 is highly expressed in cortical excitatory neurons and, reducing its expression in mice accelerates the maturation of excitatory synapses during sensitive developmental periods, restricts the critical period window for plasticity, and impairs cognition. However, its specific role in interneurons remains largely undetermined. In this study, we investigated the effects of conditional Syngap1 disruption in medial ganglionic eminence (MGE)-derived interneurons on hippocampal interneuron firing properties and excitatory synaptic inputs, as well as on pyramidal cell synaptic inhibition and synaptic integration. We show that conditional Syngap1 disruption in MGE-derived interneurons results in cell-specific impairment of firing properties of hippocampal Nkx2.1 fast-spiking interneurons, with enhancement of their AMPA receptor (AMPAR)-mediated excitatory synaptic inputs but compromised short-term plasticity. In contrast, regular-spiking Nkx2.1 interneurons are largely unaffected. These changes are associated with impaired pyramidal cell synaptic inhibition and enhanced summation of excitatory responses. Unexpectedly, we found that the Syngap1flox allele used in this study contains inverted loxP sites and that its targeted recombination in MGE-derived interneurons induces some cell loss during embryonic development and the reversible inversion of the sequence flanked by the loxP sites in postmitotic cells. Together, these results suggest that Syngap1 plays a role in cell-specific regulation of hippocampal interneuron function and inhibition of pyramidal cells in mice. However, because of our finding that the Syngap1flox allele used in this study contains inverted loxP sites, it will be important to further investigate interneuron function using a different Syngap1 conditional allele.

MED13L and its disease-associated variants influence the dendritic development of cerebral cortical neurons in the mammalian brain.

The mediator complex comprises multiple subcellular subunits that collectively function as a molecular interface between RNA polymerase II and gene-specific transcription factors. Recently, genetic variants to one subunit of the complex, known as MED13L (mediator complex subunit 13 like), have been implicated in syndromic intellectual disability and distinct facial features, frequently accompanied by congenital heart defects. We investigated the impact of five disease-associated MED13L variants on the subcellular localization and biochemical stability of MED13L protein in vitro and in vivo. In overexpression assays using cortical neurons from embryonic mouse cerebral cortices transduced by in utero electroporation-mediated gene transfer, we found that mouse orthologues of human MED13L-p.P866L and -p.T2162M missense variants accumulated in the nucleus, while the p.S2163L and p.S2177Y variants were diffusely distributed in the cytoplasm. In contrast, we found that the p.Q1922* truncation variant was barely detectable in transduced cells, a phenotype reminiscent of this variant that results in MED13L haploinsufficiency in humans. Next, we analyzed these variants for their effects on neuronal migration, dendritic growth, spine morphology, and axon elongation of cortical neurons in vivo. There, we found that overexpression of the p.P866L variant resulted in reduced number and length of dendrites of cortical layer II/III pyramidal neurons. Furthermore, we show that mMED13L-knockdown abrogated dendritic growth in vivo, and this effect was significantly rescued by co-electroporation of an RNAi-resistant mMED13L, but weakly by the p.T2162M variant, and not at all by the p.S2163L variant. However, overexpression of the p.S2163L variant inhibited mature dendritic spine formation in vivo. Expression of each of the 5 variants did not affect neuronal cell migration and callosal axon elongation in vivo. Taken together, our results demonstrate that MED13L expression is relevant to corticogenesis and influences the dendritic branching characteristics of cortical excitatory neurons. Our study also suggests that disease-associated MED13L variants may directly cause morphological and functional defects in cortical neurons in different ways.

Abnormal brain state distribution and network connectivity in a SYNGAP1 rat model.

Mutations in the SYNGAP1 gene are one of the common predictors of neurodevelopmental disorders, commonly resulting in individuals developing autism, intellectual disability, epilepsy, and sleep deficits. EEG recordings in neurodevelopmental disorders show potential to identify clinically translatable biomarkers to both diagnose and track the progress of novel therapeutic strategies, as well as providing insight into underlying pathological mechanisms. In a rat model of SYNGAP1 haploinsufficiency in which the exons encoding the calcium/lipid binding and GTPase-activating protein domains have been deleted (Syngap+/Δ-GAP ), we analysed the duration and occurrence of wake, non-rapid eye movement and rapid eye movement brain states during 6 h multi-electrode EEG recordings. We find that although Syngap+/Δ-GAP animals spend an equivalent percent time in wake and sleep states, they have an abnormal brain state distribution as the number of wake and non-rapid eye movement bouts are reduced and there is an increase in the average duration of both wake and non-rapid eye movement epochs. We perform connectivity analysis by calculating the average imaginary coherence between electrode pairs at varying distance thresholds during these states. In group averages from pairs of electrodes at short distances from each other, a clear reduction in connectivity during non-rapid eye movement is present between 11.5 Hz and 29.5 Hz, a frequency range that overlaps with sleep spindles, oscillatory phenomena thought to be important for normal brain function and memory consolidation. Sleep abnormalities were mostly uncorrelated to the electrophysiological signature of absence seizures, spike and wave discharges, as was the imaginary coherence deficit. Sleep spindles occurrence, amplitude, power and spread across multiple electrodes were not reduced in Syngap+/Δ-GAP rats, with only a small decrease in duration detected. Nonetheless, by analysing the dynamic imaginary coherence during sleep spindles, we found a reduction in high-connectivity instances between short-distance electrode pairs. Finally comparing the dynamic imaginary coherence during sleep spindles between individual electrode pairs, we identified a group of channels over the right somatosensory, association and visual cortices that have a significant reduction in connectivity during sleep spindles in mutant animals. This matched a significant reduction in connectivity during spindles when averaged regional comparisons were made. These data suggest that Syngap+/Δ-GAP rats have altered brain state dynamics and EEG connectivity, which may have clinical relevance for SYNGAP1 haploinsufficiency in humans.

PD15-01 KANK1 GENETIC AND GENOMIC ANOMALIES AFFECT GENITOURINARY (GU) DEVELOPMENT IN HUMANS

You have accessJournal of UrologyPediatric Urology I (PD15)1 Sep 2021PD15-01 KANK1 GENETIC AND GENOMIC ANOMALIES AFFECT GENITOURINARY (GU) DEVELOPMENT IN HUMANS Marisol O'Neill, and Dolores Lamb Marisol O'NeillMarisol O'Neill More articles by this author , and Dolores LambDolores Lamb More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001997.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: 9p24.3 is a chromosome hotspot for human GU anomalies with micro-deletions or -duplications associated with ambiguous genitalia and gonadal dysgenesis. One gene in 9p24.3, DMRT1, is needed for normal testicular fetal development. Using array comparative genomic hybridization (aCGH) together with retrospective literature/database analysis, locus mapping identified 60 patients with 9p24.3 CNVs of 37 duplications and 23 microdeletions. 50 of these patients had disorders of sexual differentiation, 7 had congenital anomalies of the kidney and urinary tract (CAKUT). We identified a second gene candidate in the minimal overlap region of 9p24.3 5' to DMRT1 that encompasses KN Motif and Ankyrin Repeat Domain Containing Protein 1 (KANK1). We used human and mouse models to test the hypothesis that genetic and genomic defects of KANK1 also resulted in anomalies of the external genitalia and CAKUT. METHODS: A mouse model of haploinsufficiency was generated using CRISPR-Cas9 to delete Kank1 and mice phenotyped. Penile and renal anomalies were defined by micro-CT analysis. Patients with upper and lower tract genitourinary birth defects underwent phenotypic analysis, genetic and genomic analysis by aCGH and whole exome sequencing. RESULTS: A mouse model of Kank1 haploinsufficiency exhibited renal dysfunction, penile anomalies and subfertility. KANK1 genetic variants were identified by Whole Exome Sequencing (WES) in patients with GU anomalies-2 had novel variants; one individual with an absent cervix, vaginal atresia and two novel variants resulting in nonsynonymous changes of two consecutive amino acids (p.C1187F and p.N118Y) both predicted to be deleterious. 14 individuals were identified with 10 rare variants, five of which (rs61737971, rs148744436, rs143675911, rs74458120, rs370051574) were present in individuals with CAKUT, renal insufficiency, focal segmental glomerulosclerosis, abnormal reproductive track development, hypospadias, bladder exstrophy- glomerular fibrosis, renal dysfunction, penile anomalies were also present in the Kank1 haploinsufficient mouse models. CONCLUSIONS: This study identifies a novel mechanism that underlies some GU birth defects which are usually surgically repaired with the etiology defined as idiopathic. This study identified renal dysfunction and a potentially severe kidney phenotype, as well as developmental anomalies of the penis caused by KANK1 haploinsufficiency in humans & mouse models. Source of Funding: T32 DK007763 (MO was a trainee) & R01DK078121 from NIDDK & Frederick J. and Theresa Dow Wallace Fund of the NY Community Trust (awarded to DJL) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e272-e272 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Marisol O'Neill More articles by this author Dolores Lamb More articles by this author Expand All Advertisement Loading ...

Gata2 heterozygous mutant mice exhibit reduced inflammatory responses and impaired bacterial clearance

SummaryInfectious diseases continually pose global medical challenges. The transcription factor GATA2 establishes gene networks and defines cellular identity in hematopoietic stem/progenitor cells and in progeny committed to specific lineages. GATA2-haploinsufficient patients exhibit a spectrum of immunodeficiencies associated with bacterial, viral, and fungal infections. Despite accumulating clinical knowledge of the consequences of GATA2 haploinsufficiency in humans, it is unclear how GATA2 haploinsufficiency compromises host anti-infectious defenses. To address this issue, we examined Gata2-heterozygous mutant (G2Het) mice as a model for human GATA2 haploinsufficiency. In vivo inflammation imaging and cytokine multiplex analysis demonstrated that G2Het mice had attenuated inflammatory responses with reduced levels of inflammatory cytokines, particularly IFN-γ, IL-12p40, and IL-17A, during lipopolysaccharide-induced acute inflammation. Consequently, bacterial clearance was significantly impaired in G2Het mice after cecal ligation and puncture-induced polymicrobial peritonitis. These results provide direct molecular insights into GATA2-directed host defenses and the pathogenic mechanisms underlying observed immunodeficiencies in GATA2-haploinsufficient patients.

Loss of function MPZ mutation causes milder CMT1B neuropathy.

Mutations in Myelin Protein Zero (MPZ) cause CMT1B, the second leading cause of CMT1. Many of the >200 mutations cause neuropathy through a toxic gain of function by the mutant protein such as ER retention, activation of the Unfolded Protein Response (UPR) or disruption of myelin compaction. While there is extensive literature on the loss of function consequences of MPZ in heterozygous Mpz +/- null mice, there is little known of the consequences of MPZ haploinsufficiency in humans. We identified six patients from different families with p.Tyr68Ter or p.Asp104fs heterozygous mutations of MPZ that are predicted to cause a premature termination and nonsense mediated decay of the mutant allele. Five patients were evaluated in Milan and one in Iowa City; all should be haploinsufficient for MPZ. Patients were evaluated clinically and by electrophysiology. Sensory ataxia dominated the clinical presentation with only mild weakness present in five of the six patients. Symptoms presented in adulthood in all patients and only one individual had a CMTNSv2 >5. Deep tendon reflexes were absent in all patients. Patients with likely MPZ loss of function due to mutations that cause haplodeficiency in MPZ have a mild, predominantly large fiber sensory neuropathy that serves as a human equivalent to the neuropathy observed in heterozygous Mpz null mice. Successful therapeutic approaches in treating Mpz deficient mice may be candidates for trials in these and similar patients.

Haploinsufficiency of the Sin3/HDAC corepressor complex member SIN3B causes a syndromic intellectual disability/autism spectrum disorder

Proteins involved in transcriptional regulation harbor a demonstrated enrichment of mutations in neurodevelopmental disorders. The Sin3 (Swi-independent 3)/histone deacetylase (HDAC) complex plays a central role in histone deacetylation and transcriptional repression. Among the two vertebrate paralogs encoding the Sin3 complex, SIN3A variants cause syndromic intellectual disability, but the clinical consequences of SIN3B haploinsufficiency in humans are uncharacterized. Here, we describe a syndrome hallmarked by intellectual disability, developmental delay, and dysmorphic facial features with variably penetrant autism spectrum disorder, congenital malformations, corpus callosum defects, and impaired growth caused by disruptive SIN3B variants. Using chromosomal microarray or exome sequencing, and through international data sharing efforts, we identified nine individuals with heterozygous SIN3B deletion or single-nucleotide variants. Five individuals harbor heterozygous deletions encompassing SIN3B that reside within a ∼230 kb minimal region of overlap on 19p13.11, two individuals have a rare nonsynonymous substitution, and two individuals have a single-nucleotide deletion that results in a frameshift and predicted premature termination codon. To test the relevance of SIN3B impairment to measurable aspects of the human phenotype, we disrupted the orthologous zebrafish locus by genome editing and transient suppression. The mutant and morphant larvae display altered craniofacial patterning, commissural axon defects, and reduced body length supportive of an essential role for Sin3 function in growth and patterning of anterior structures. To investigate further the molecular consequences of SIN3B variants, we quantified genome-wide enhancer and promoter activity states by using H3K27ac ChIP-seq. We show that, similar to SIN3A mutations, SIN3B disruption causes hyperacetylation of a subset of enhancers and promoters in peripheral blood mononuclear cells. Together, these data demonstrate that SIN3B haploinsufficiency leads to a hitherto unknown intellectual disability/autism syndrome, uncover a crucial role of SIN3B in the central nervous system, and define the epigenetic landscape associated with Sin3 complex impairment.

OR02-01 Gene Dosage Changes in RBFOX-2 Can Affect Upper and Lower Tract Genitourinary Development

Searching for genetic and genomic alterations in patients with genitourinary (GU) birth defects can aid in the identification of genes that are important for normal GU development. Using data in DECIPHER, RBFOX-2, located at 22q12 was identified as one such candidate gene and the effect of haploinsufficiency in humans and a mouse model (Rbfox-2α/+) assessed. RBFOX2 is an RNA-binding protein regulating alternative splicing. It also impacts steroid receptor transcriptional activity. RBFOX family members are thought to affect different developmental programs via isoform-specific localization and splicing activities. RBFOX-2 plays a vital role in multiple developmental processes including heart, brain, muscle and testis development. “Non-syndromic” patients with congenital GU anomalies were more likely to harbor RBFOX-2 CNVs (2.4%; hypospadias, 21% cryptorchidism, 29% vesicoureteral reflux and 29% ureteropelvic junction obstruction) relative to the general population (0.11%) indicating that 22q12 is a hotspot genetic locus for GU anomalies. CNVs (microdeletions and microduplications) spanning RBFOX-2 were identified in twenty-seven humans with a wide range of birth defects(intellectual disability, CNS, cardiac, genitourinary, facial, hearing, vision, growth and stature anomalies) and the phenotype of the haploinsufficient Rbfox-2α/+mouse model closely correlated with the human phenotype, although Rbfox-2α/+ andRbfox-2α/α are neonatal lethal. By E18.5 day of development 100% of the Rbfox-2α/+ and null fetuses exhibited hypospadias, megacystis (50%) and bilateral hydroureteronephrosis. RNA Seq was performed on genital tubercles from three Rbfox2fl/fl, Gdf9 icre+ and three Rbfox2fl/fl P1 males. 377 genes were differentially expressed between the two groups including 212 genes which were upregulated and 165 genes which were downregulated. Hallmark gene set analysis revealed downregulation of Wnt/β-catenin signaling and hedgehog signaling, both pathways which are well defined in genital development. Gene ontology (GO) analysis further defined downregulation of pathways (fibroblast growth factor, retinoic, androgen, estrogen and G-protein receptor signaling) including those involving negative regulation of embryonic development, epithelial development, morphogenesis of epithelial tube, renal, ureter, limb and several other developmental pathways. These results support the hypothesis that loss of Rbfox2 during development leads to hypospadias through impaired development of the urethral epithelium.

Characterization of SETD1A haploinsufficiency in humans and Drosophila defines a novel neurodevelopmental syndrome.

Defects in histone methyltransferases (HMTs) are major contributing factors in neurodevelopmental disorders (NDDs). Heterozygous variants of SETD1A involved in histone H3 lysine 4 (H3K4) methylation were previously identified in individuals with schizophrenia. Here, we define the clinical features of the Mendelian syndrome associated with haploinsufficiency of SETD1A by investigating 15 predominantly pediatric individuals who all have de novo SETD1A variants. These individuals present with a core set of symptoms comprising global developmental delay and/or intellectual disability, subtle facial dysmorphisms, behavioral and psychiatric problems. We examined cellular phenotypes in three patient-derived lymphoblastoid cell lines with three variants: p.Gly535Alafs*12, c.4582-2_4582delAG, and p.Tyr1499Asp. These patient cell lines displayed DNA damage repair defects that were comparable to previously observed RNAi-mediated depletion of SETD1A. This suggested that these variants, including the p.Tyr1499Asp in the catalytic SET domain, behave as loss-of-function (LoF) alleles. Previous studies demonstrated a role for SETD1A in cell cycle control and differentiation. However, individuals with SETD1A variants do not show major structural brain defects or severe microcephaly, suggesting that defective proliferation and differentiation of neural progenitors is unlikely the single underlying cause of the disorder. We show here that the Drosophila melanogaster SETD1A orthologue is required in postmitotic neurons of the fly brain for normal memory, suggesting a role in post development neuronal function. Together, this study defines a neurodevelopmental disorder caused by dominant de novo LoF variants in SETD1A and further supports a role for H3K4 methyltransferases in the regulation of neuronal processes underlying normal cognitive functioning.

Transcription and Beyond: Delineating FOXG1 Function in Cortical Development and Disorders.

Forkhead Box G1 (FOXG1) is a member of the Forkhead family of genes with non-redundant roles in brain development, where alteration of this gene’s expression significantly affects the formation and function of the mammalian cerebral cortex. FOXG1 haploinsufficiency in humans is associated with prominent differences in brain size and impaired intellectual development noticeable in early childhood, while homozygous mutations are typically fatal. As such, FOXG1 has been implicated in a wide spectrum of congenital brain disorders, including the congenital variant of Rett syndrome, infantile spasms, microcephaly, autism spectrum disorder (ASD) and schizophrenia. Recent technological advances have yielded greater insight into phenotypic variations observed in FOXG1 syndrome, molecular mechanisms underlying pathogenesis of the disease, and multifaceted roles of FOXG1 expression. In this review, we explore the emerging mechanisms of FOXG1 in a range of transcriptional to posttranscriptional events in order to evolve our current view of how a single transcription factor governs the assembly of an elaborate cortical circuit responsible for higher cognitive functions and neurological disorders.

Heterozygosity for Nuclear Factor One X in mice models features of Malan syndrome.

BackgroundNuclear Factor One X (NFIX) haploinsufficiency in humans results in Malan syndrome, a disorder characterized by overgrowth, macrocephaly and intellectual disability. Although clinical assessments have determined the underlying symptomology of Malan syndrome, the fundamental mechanisms contributing to the enlarged head circumference and intellectual disability in these patients remains undefined.MethodsHere, we used Nfix heterozygous mice as a model to investigate these aspects of Malan syndrome. Volumetric magnetic resonance imaging (MRI) was used to calculate the volumes of 20 brain sub regions. Diffusion tensor MRI was used to perform tractography-based analyses of the corpus callosum, hippocampal commissure, and anterior commissure, as well as structural connectome mapping of the whole brain. Immunohistochemistry examined the neocortical cellular populations. Two behavioral assays were performed, including the active place avoidance task to assess spatial navigation and learning and memory function, and the 3-chambered sociability task to examine social behaviour.FindingsAdult Nfix+/− mice exhibit significantly increased brain volume (megalencephaly) compared to wildtypes, with the cerebral cortex showing the highest increase. Moreover, all three forebrain commissures, in particular the anterior commissure, revealed significantly reduced fractional anisotropy, axial and radial diffusivity, and tract density intensity. Structural connectome analyses revealed aberrant connectivity between many crucial brain regions. Finally, Nfix+/− mice exhibit behavioral deficits that model intellectual disability.InterpretationCollectively, these data provide a significant conceptual advance in our understanding of Malan syndrome by suggesting that megalencephaly underlies the enlarged head size of these patients, and that disrupted cortical connectivity may contribute to the intellectual disability these patients exhibit.FundAustralian Research Council (ARC) Discovery Project Grants, ARC Fellowship, NYSTEM and Australian Postgraduate Fellowships.

SYNGAP1 heterozygosity disrupts sensory processing by reducing touch-related activity within somatosensory cortex circuits.

In addition to cognitive impairments, neurodevelopmental disorders (NDDs) often result in sensory processing deficits. However, the biological mechanisms that underlie impaired sensory processing associated with NDDs are generally understudied and poorly understood. We found that SYNGAP1 haploinsufficiency in humans, which causes a sporadic neurodevelopmental disorder defined by cognitive impairment, autistic features, and epilepsy, also leads to deficits in tactile-related sensory processing. In vivo neurophysiological analysis in Syngap1 mouse models revealed that upper-lamina neurons in somatosensory cortex (SSC) weakly encode information related to touch. This was caused by reduced synaptic connectivity and impaired intrinsic excitability within upper-lamina SSC neurons. These results were unexpected given that Syngap1 heterozygosity is known to cause circuit hyperexcitability in brain areas more directly linked to cognitive functions. Thus, Syngap1 heterozygosity causes a range of circuit-specific pathologies, including reduced activity within cortical neurons required for touch processing, which may contribute to sensory phenotypes observed in patients.

Highly specific monoclonal antibodies for allosteric inhibition and immunodetection of the human pancreatic zinc transporter ZnT8

Solute carrier family 30 member 8 (SLC30A8), encoding the pancreatic zinc transporter ZnT8, is a susceptibility gene for type 2 diabetes (T2D). Reducing ZnT8 transport activity or down-regulating its cellular expression is hypothesized to be an antidiabetogenic strategy mimicking the protective effect of SLC30A8 haploinsufficiency in humans. However, research tools to inhibit ZnT8 activity and measure cellular ZnT8 levels are not available. Here, we report the identification of two anti-ZnT8 mAbs applicable to addressing these unmet needs. Both mAbs exhibited subnanomolar affinities for human ZnT8 and were selective against homologous zinc transporters with distinct cross-species reactivities and epitope recognition. We showed that antigen-binding fragments (Fabs) protected ZnT8 from unfolding and inhibited ZnT8-mediated zinc transport in proteoliposomes. Negative-stain EM revealed a ternary binding complex of a ZnT8 monomer and two different Fabs at a 1:1:1 stoichiometry. Moreover, dual bindings of two different mAbs to a single ZnT8 protein multiplied the individual anti-ZnT8 specificities, enabling quantification of cellular ZnT8 levels by homogeneous time-resolved fluorescence (HTRF). Our results demonstrate the utilities of the two generated mAbs as allosteric inhibitors and highly specific biosensors of human ZnT8.

Genetic variants help define the role of the MC4R C-terminus in signaling and cell surface stability

Screening 92,445 subjects in the Geisinger-Regeneron DiscovEHR cohort, we identified 5 patients heterozygous for nonsense mutations causing early terminations at Glu307 or Leu328 on the C-terminus of melanocortin 4 receptor (MC4R). Two Q307Ter carriers are severely obese (BMI > 40), while one is overweight (BMI > 25). One L328Ter carrier is overweight and the other is lean. Pedigree analysis for two Q307Ter carriers shows segregation of the variant with higher BMI. Functionally, MC4R(Q307Ter) eliminated receptor surface expression and signaling, while MC4R(L328Ter) functioned like the wild-type receptor. MC4R(Q307Ter) is therefore a loss of function (LOF) variant and the region between the two truncation sites identified in our patients is critical to MC4R function. Truncating MC4R at various C-terminal positions between these two variant sites, we find that cysteine318 sits at a critical junction for receptor trafficking and function. We show that MC4R is lipid modified at cysteine318 and cysteine319. Therefore, truncation early in the MC4R C-terminus results in haploinsufficiency in humans while truncation after the first lipid-modification site is well tolerated. MC4R haploinsufficiency clearly segregates with higher BMI; however, severe obesity is not fully penetrant even in MC4R LOF carriers, suggesting critical roles for environmental and lifestyle factors in MC4R monogenic obesity.

Long telomeres protect against age-dependent cardiac disease caused by NOTCH1 haploinsufficiency.

Diseases caused by gene haploinsufficiency in humans commonly lack a phenotype in mice that are heterozygous for the orthologous factor, impeding the study of complex phenotypes and critically limiting the discovery of therapeutics. Laboratory mice have longer telomeres relative to humans, potentially protecting against age-related disease caused by haploinsufficiency. Here, we demonstrate that telomere shortening in NOTCH1-haploinsufficient mice is sufficient to elicit age-dependent cardiovascular disease involving premature calcification of the aortic valve, a phenotype that closely mimics human disease caused by NOTCH1 haploinsufficiency. Furthermore, progressive telomere shortening correlated with severity of disease, causing cardiac valve and septal disease in the neonate that was similar to the range of valve disease observed within human families. Genes that were dysregulated due to NOTCH1 haploinsufficiency in mice with shortened telomeres were concordant with proosteoblast and proinflammatory gene network alterations in human NOTCH1 heterozygous endothelial cells. These dysregulated genes were enriched for telomere-contacting promoters, suggesting a potential mechanism for telomere-dependent regulation of homeostatic gene expression. These findings reveal a critical role for telomere length in a mouse model of age-dependent human disease and provide an in vivo model in which to test therapeutic candidates targeting the progression of aortic valve disease.