RNA variant assessment using transactivation and transdifferentiation.

Understanding the impact of splicing and nonsense variants on RNA is crucial for the resolution of variant classification as well as their suitability for precision medicine interventions. This is primarily enabled through RNA studies involving transcriptomics followed by targeted assays using RNA isolated from clinically accessible tissues (CATs) such as blood or skin of affected individuals. Insufficient disease gene expression in CATs does however pose a major barrier to RNA based investigations, which we show is relevant to 1,436 Mendelian disease genes. We term these "silent" Mendelian genes (SMGs), the largest portion (36%) of which are associated with neurological disorders. We developed two approaches to induce SMG expression in human dermal fibroblasts (HDFs) to overcome this limitation, including CRISPR-activation-based gene transactivation and fibroblast-to-neuron transdifferentiation. Initial transactivation screens involving 40 SMGs stimulated our development of a highly multiplexed transactivation system culminating in the 6- to 90,000-fold induction of expression of 20/20 (100%) SMGs tested in HDFs. Transdifferentiation of HDFs directly to neurons led to expression of 193/516 (37.4%) of SMGs implicated in neurological disease. The magnitude and isoform diversity of SMG expression following either transactivation or transdifferentiation was comparable to clinically relevant tissues. We apply transdifferentiation and/or gene transactivation combined with short- and long-read RNA sequencing to investigate the impact that variants in USH2A, SCN1A, DMD, and PAK3 have on RNA using HDFs derived from affected individuals. Transactivation and transdifferentiation represent rapid, scalable functional genomic solutions to investigate variants impacting SMGs in the patient cell and genomic context.

Proteomic analysis of the developing mammalian brain links PCDH19 to the Wnt/ß-catenin signalling pathway.

Clustering Epilepsy (CE) is a neurological disorder caused by pathogenic variants of the Protocadherin 19 (PCDH19) gene. PCDH19 encodes a protein involved in cell adhesion and Estrogen Receptor α mediated-gene regulation. To gain further insights into the molecular role of PCDH19 in the brain, we investigated the PCDH19 interactome in the developing mouse hippocampus and cortex. Combined with a meta-analysis of all reported PCDH19 interacting proteins, our results show that PCDH19 interacts with proteins involved in actin, microtubule, and gene regulation. We report CAPZA1, αN-catenin and, importantly, ß-catenin as novel PCDH19 interacting proteins. Furthermore, we show that PCDH19 is a regulator of ß-catenin transcriptional activity, and that this pathway is disrupted in CE individuals. Overall, our results support the involvement of PCDH19 in the cytoskeletal network and point to signalling pathways where PCDH19 plays critical roles.

A synonymous UPF3B variant causing a speech disorder implicates NMD as a regulator of neurodevelopmental disorder gene networks.

Loss-of-function mutations of the X-chromosome gene UPF3B cause male neurodevelopmental disorders (NDDs) via largely unknown mechanisms. We investigated initially by interrogating a novel synonymous UPF3B variant in a male with absent speech. In silico and functional studies using cell lines derived from this individual show altered UPF3B RNA splicing. The resulting mRNA species encodes a frame-shifted protein with a premature termination codon (PTC) predicted to elicit degradation via nonsense-mediated mRNA decay (NMD). UPF3B mRNA was reduced in the cell line, and no UPF3B protein was produced, confirming a loss-of-function allele. UPF3B is itself involved in the NMD mechanism which degrades both PTC-bearing mutant transcripts and also many physiological transcripts. RNAseq analysis showed that ~1.6% of mRNAs exhibited altered expression. These mRNA changes overlapped and correlated with those we identified in additional cell lines obtained from individuals harbouring other UPF3B mutations, permitting us to interrogate pathogenic mechanisms of UPF3B-associated NDDs. We identified 102 genes consistently deregulated across all UPF3B mutant cell lines. Of the 51 upregulated genes, 75% contained an NMD-targeting feature, thus identifying high-confidence direct NMD targets. Intriguingly, 22 of the dysregulated genes encoded known NDD genes, suggesting UPF3B-dependent NMD regulates gene networks critical for cognition and behaviour. Indeed, we show that 78.5% of all NDD genes encode a transcript predicted to be targeted by NMD. These data describe the first synonymous UPF3B mutation in a patient with prominent speech and language disabilities and identify plausible mechanisms of pathology downstream of UPF3B mutations involving the deregulation of NDD-gene networks.

Genomic and phenotypic characterization of 404 individuals with neurodevelopmental disorders caused by CTNNB1 variants.

PURPOSE: Germline loss-of-function variants in CTNNB1 cause neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV; OMIM 615075) and are the most frequent, recurrent monogenic cause of cerebral palsy (CP). We investigated the range of clinical phenotypes owing to disruptions of CTNNB1 to determine the association between NEDSDV and CP. METHODS: Genetic information from 404 individuals with collectively 392 pathogenic CTNNB1 variants were ascertained for the study. From these, detailed phenotypes for 52 previously unpublished individuals were collected and combined with 68 previously published individuals with comparable clinical information. The functional effects of selected CTNNB1 missense variants were assessed using TOPFlash assay. RESULTS: The phenotypes associated with pathogenic CTNNB1 variants were similar. A diagnosis of CP was not significantly associated with any set of traits that defined a specific phenotypic subgroup, indicating that CP is not additional to NEDSDV. Two CTNNB1 missense variants were dominant negative regulators of WNT signaling, highlighting the utility of the TOPFlash assay to functionally assess variants. CONCLUSION: NEDSDV is a clinically homogeneous disorder irrespective of initial clinical diagnoses, including CP, or entry points for genetic testing.

Abnormal Behavior and Cortical Connectivity Deficits in Mice Lacking Usp9x.

Genetic association studies have identified many factors associated with neurodevelopmental disorders such as autism spectrum disorder (ASD). However, the way these genes shape neuroanatomical structure and connectivity is poorly understood. Recent research has focused on proteins that act as points of convergence for multiple factors, as these may provide greater insight into understanding the biology of neurodevelopmental disorders. USP9X, a deubiquitylating enzyme that regulates the stability of many ASD-related proteins, is one such point of convergence. Loss of function variants in human USP9X lead to brain malformations, which manifest as a neurodevelopmental syndrome that frequently includes ASD, but the underlying structural and connectomic abnormalities giving rise to patient symptoms is unknown. Here, we analyzed forebrain-specific Usp9x knockout mice (Usp9x-/y) to address this knowledge gap. Usp9x-/y mice displayed abnormal communication and social interaction behaviors. Moreover, the absence of Usp9x culminated in reductions to the size of multiple brain regions. Diffusion tensor magnetic resonance imaging revealed deficits in all three major forebrain commissures, as well as long-range hypoconnectivity between cortical and subcortical regions. These data identify USP9X as a key regulator of brain formation and function, and provide insights into the neurodevelopmental syndrome arising as a consequence of USP9X mutations in patients.

Integrated in silico and experimental assessment of disease relevance of PCDH19 missense variants.

PCDH19 is a nonclustered protocadherin molecule involved in axon bundling, synapse function, and transcriptional coregulation. Pathogenic variants in PCDH19 cause infantile-onset epilepsy known as PCDH19-clustering epilepsy or PCDH19-CE. Recent advances in DNA-sequencing technologies have led to a significant increase in the number of reported PCDH19-CE variants, many of uncertain significance. We aimed to determine the best approaches for assessing the disease relevance of missense variants in PCDH19. The application of the American College of Medical Genetics and Association for Molecular Pathology (ACMG-AMP) guidelines was only 50% accurate. Using a training set of 322 known benign or pathogenic missense variants, we identified MutPred2, MutationAssessor, and GPP as the best performing in silico tools. We generated a protein structural model of the extracellular domain and assessed 24 missense variants. We also assessed 24 variants using an in vitro reporter assay. A combination of these tools was 93% accurate in assessing known pathogenic and benign PCDH19 variants. We increased the accuracy of the ACMG-AMP classification of 45 PCDH19 variants from 50% to 94%, using these tools. In summary, we have developed a robust toolbox for the assessment of PCDH19 variant pathogenicity to improve the accuracy of PCDH19-CE variant classification.

De Novo Loss-of-Function Mutations in USP9X Cause a Female-Specific Recognizable Syndrome with Developmental Delay and Congenital Malformations.

Mutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affected females are also reported in known ciliopathy syndromes, we examined the role of USP9X in the primary cilium and found that endogenous USP9X localizes along the length of the ciliary axoneme, indicating that its loss of function could indeed disrupt cilium-regulated processes. Absence of dysregulated ciliary parameters in affected female-derived fibroblasts, however, points toward spatiotemporal specificity of ciliary USP9X (dys-)function.

PCDH19 regulation of neural progenitor cell differentiation suggests asynchrony of neurogenesis as a mechanism contributing to PCDH19 Girls Clustering Epilepsy.

PCDH19-Girls Clustering Epilepsy (PCDH19-GCE) is a childhood epileptic encephalopathy characterised by a spectrum of neurodevelopmental problems. PCDH19-GCE is caused by heterozygous loss-of-function mutations in the X-chromosome gene, Protocadherin 19 (PCDH19) encoding a cell-cell adhesion molecule. Intriguingly, hemizygous males are generally unaffected. As PCDH19 is subjected to random X-inactivation, heterozygous females are comprised of a mosaic of cells expressing either the normal or mutant allele, which is thought to drive pathology. Despite being the second most prevalent monogeneic cause of epilepsy, little is known about the role of PCDH19 in brain development. In this study we show that PCDH19 is highly expressed in human neural stem and progenitor cells (NSPCs) and investigate its function in vitro in these cells of both mouse and human origin. Transcriptomic analysis of mouse NSPCs lacking Pcdh19 revealed changes to genes involved in regulation of neuronal differentiation, and we subsequently show that loss of Pcdh19 causes increased NSPC neurogenesis. We reprogramed human fibroblast cells harbouring a pathogenic PCDH19 mutation into human induced pluripotent stem cells (hiPSC) and employed neural differentiation of these to extend our studies into human NSPCs. As in mouse, loss of PCDH19 function caused increased neurogenesis, and furthermore, we show this is associated with a loss of human NSPC polarity. Overall our data suggests a conserved role for PCDH19 in regulating mammalian cortical neurogenesis and has implications for the pathogenesis of PCDH19-GCE. We propose that the difference in timing or "heterochrony" of neuronal cell production originating from PCDH19 wildtype and mutant NSPCs within the same individual may lead to downstream asynchronies and abnormalities in neuronal network formation, which in-part predispose the individual to network dysfunction and epileptic activity.

THOC2 Mutations Implicate mRNA-Export Pathway in X-Linked Intellectual Disability.

Export of mRNA from the cell nucleus to the cytoplasm is essential for protein synthesis, a process vital to all living eukaryotic cells. mRNA export is highly conserved and ubiquitous. Mutations affecting mRNA and mRNA processing or export factors, which cause aberrant retention of mRNAs in the nucleus, are thus emerging as contributors to an important class of human genetic disorders. Here, we report that variants in THOC2, which encodes a subunit of the highly conserved TREX mRNA-export complex, cause syndromic intellectual disability (ID). Affected individuals presented with variable degrees of ID and commonly observed features included speech delay, elevated BMI, short stature, seizure disorders, gait disturbance, and tremors. X chromosome exome sequencing revealed four missense variants in THOC2 in four families, including family MRX12, first ascertained in 1971. We show that two variants lead to decreased stability of THOC2 and its TREX-complex partners in cells derived from the affected individuals. Protein structural modeling showed that the altered amino acids are located in the RNA-binding domains of two complex THOC2 structures, potentially representing two different intermediate RNA-binding states of THOC2 during RNA transport. Our results show that disturbance of the canonical molecular pathway of mRNA export is compatible with life but results in altered neuronal development with other comorbidities.

Robust imaging and gene delivery to study human lymphoblastoid cell lines.

Lymphoblastoid cell lines (LCLs) have been by far the most prevalent cell type used to study the genetics underlying normal and disease-relevant human phenotypic variation, across personal to epidemiological scales. In contrast, only few studies have explored the use of LCLs in functional genomics and mechanistic studies. Two major reasons are technical, as (1) interrogating the sub-cellular spatial information of LCLs is challenged by their non-adherent nature, and (2) LCLs are refractory to gene transfection. Methodological details relating to techniques that overcome these limitations are scarce, largely inadequate (without additional knowledge and expertise), and optimisation has never been described. Here we compare, optimise, and convey such methods in-depth. We provide a robust method to adhere LCLs to coverslips, which maintained cellular integrity, morphology, and permitted visualisation of sub-cellular structures and protein localisation. Next, we developed the use of lentiviral-based gene delivery to LCLs. Through empirical and combinatorial testing of multiple transduction conditions, we improved transduction efficiency from 3% up to 48%. Furthermore, we established strategies to purify transduced cells, to achieve sustainable cultures containing >85% transduced cells. Collectively, our methodologies provide a vital resource that enables the use of LCLs in functional cell and molecular biology experiments. Potential applications include the characterisation of genetic variants of unknown significance, the interrogation of cellular disease pathways and mechanisms, and high-throughput discovery of genetic modifiers of disease states among others.

Seizures are regulated by ubiquitin-specific peptidase 9 X-linked (USP9X), a de-ubiquitinase.

Epilepsy is a common disabling disease with complex, multifactorial genetic and environmental etiology. The small fraction of epilepsies subject to Mendelian inheritance offers key insight into epilepsy disease mechanisms; and pathologies brought on by mutations in a single gene can point the way to generalizable therapeutic strategies. Mutations in the PRICKLE genes can cause seizures in humans, zebrafish, mice, and flies, suggesting the seizure-suppression pathway is evolutionarily conserved. This pathway has never been targeted for novel anti-seizure treatments. Here, the mammalian PRICKLE-interactome was defined, identifying prickle-interacting proteins that localize to synapses and a novel interacting partner, USP9X, a substrate-specific de-ubiquitinase. PRICKLE and USP9X interact through their carboxy-termini; and USP9X de-ubiquitinates PRICKLE, protecting it from proteasomal degradation. In forebrain neurons of mice, USP9X deficiency reduced levels of Prickle2 protein. Genetic analysis suggests the same pathway regulates Prickle-mediated seizures. The seizure phenotype was suppressed in prickle mutant flies by the small-molecule USP9X inhibitor, Degrasyn/WP1130, or by reducing the dose of fat facets a USP9X orthologue. USP9X mutations were identified by resequencing a cohort of patients with epileptic encephalopathy, one patient harbored a de novo missense mutation and another a novel coding mutation. Both USP9X variants were outside the PRICKLE-interacting domain. These findings demonstrate that USP9X inhibition can suppress prickle-mediated seizure activity, and that USP9X variants may predispose to seizures. These studies point to a new target for anti-seizure therapy and illustrate the translational power of studying diseases in species across the evolutionary spectrum.

HCFC1 loss-of-function mutations disrupt neuronal and neural progenitor cells of the developing brain.

Both gain- and loss-of-function mutations have recently implicated HCFC1 in neurodevelopmental disorders. Here, we extend our previous HCFC1 over-expression studies by employing short hairpin RNA to reduce the expression of Hcfc1 in embryonic neural cells. We show that in contrast to over-expression, loss of Hcfc1 favoured proliferation of neural progenitor cells at the expense of differentiation and promoted axonal growth of post-mitotic neurons. To further support the involvement of HCFC1 in neurological disorders, we report two novel HCFC1 missense variants found in individuals with intellectual disability (ID). One of these variants, together with three previously reported HCFC1 missense variants of unknown pathogenicity, were functionally assessed using multiple cell-based assays. We show that three out of the four variants tested result in a partial loss of HCFC1 function. While over-expression of the wild-type HCFC1 caused reduction in HEK293T cell proliferation and axonal growth of neurons, these effects were alleviated upon over-expression of three of the four HCFC1 variants tested. One of these partial loss-of-function variants disrupted a nuclear localization sequence and the resulting protein displayed reduced ability to localize to the cell nucleus. The other two variants displayed negative effects on the expression of the HCFC1 target gene MMACHC, which is responsible for the metabolism of cobalamin, suggesting that these individuals may also be susceptible to cobalamin deficiency. Together, our work identifies plausible cellular consequences of missense HCFC1 variants and identifies likely and relevant disease mechanisms that converge on embryonic stages of brain development.

Homozygous mutation of STXBP5L explains an autosomal recessive infantile-onset neurodegenerative disorder.

We report siblings of consanguineous parents with an infantile-onset neurodegenerative disorder manifesting a predominant sensorimotor axonal neuropathy, optic atrophy and cognitive deficit. We used homozygosity mapping to identify an ∼12-Mbp interval identical by descent (IBD) between the affected individuals on chromosome 3q13.13-21.1 with an LOD score of 2.31. We combined family-based whole-exome and whole-genome sequencing of parents and affected siblings and, after filtering of likely non-pathogenic variants, identified a unique missense variant in syntaxin-binding protein 5-like (STXBP5L c.3127G>A, p.Val1043Ile [CCDS43137.1]) in the IBD interval. Considering other modes of inheritance, we also found compound heterozygous variants in FMNL3 (c.114G>C, p.Phe38Leu and c.1372T>G, p.Ile458Leu [CCDS44874.1]) located on chromosome 12. STXBP5L (or Tomosyn-2) is expressed in the central and peripheral nervous system and is known to inhibit neurotransmitter release through inhibition of the formation of the SNARE complexes between synaptic vesicles and the plasma membrane. FMNL3 is expressed more widely and is a formin family protein that is involved in the regulation of cell morphology and cytoskeletal organization. The STXBP5L p.Val1043Ile variant enhanced inhibition of exocytosis in comparison with wild-type (WT) STXBP5L. Furthermore, WT STXBP5L, but not variant STXBP5L, promoted axonal outgrowth in manipulated mouse primary hippocampal neurons. However, the FMNL3 p.Phe38Leu and p.Ile458Leu variants showed minimal effects in these cells. Collectively, our clinical, genetic and molecular data suggest that the IBD variant in STXBP5L is the likely cause of the disorder.

Increased STAG2 dosage defines a novel cohesinopathy with intellectual disability and behavioral problems.

Next generation genomic technologies have made a significant contribution to the understanding of the genetic architecture of human neurodevelopmental disorders. Copy number variants (CNVs) play an important role in the genetics of intellectual disability (ID). For many CNVs, and copy number gains in particular, the responsible dosage-sensitive gene(s) have been hard to identify. We have collected 18 different interstitial microduplications and 1 microtriplication of Xq25. There were 15 affected individuals from 6 different families and 13 singleton cases, 28 affected males in total. The critical overlapping region involved the STAG2 gene, which codes for a subunit of the cohesin complex that regulates cohesion of sister chromatids and gene transcription. We demonstrate that STAG2 is the dosage-sensitive gene within these CNVs, as gains of STAG2 mRNA and protein dysregulate disease-relevant neuronal gene networks in cells derived from affected individuals. We also show that STAG2 gains result in increased expression of OPHN1, a known X-chromosome ID gene. Overall, we define a novel cohesinopathy due to copy number gain of Xq25 and STAG2 in particular.

Mutations in USP9X are associated with X-linked intellectual disability and disrupt neuronal cell migration and growth.

With a wealth of disease-associated DNA variants being recently reported, the challenges of providing their functional characterization are mounting. Previously, as part of a large systematic resequencing of the X chromosome in 208 unrelated families with nonsyndromic X-linked intellectual disability, we identified three unique variants (two missense and one protein truncating) in USP9X. To assess the functional significance of these variants, we took advantage of the Usp9x knockout mouse we generated. Loss of Usp9x causes reduction in both axonal growth and neuronal cell migration. Although overexpression of wild-type human USP9X rescued these defects, all three USP9X variants failed to rescue axonal growth, caused reduced USP9X protein localization in axonal growth cones, and (in 2/3 variants) failed to rescue neuronal cell migration. Interestingly, in one of these families, the proband was subsequently identified to have a microdeletion encompassing ARID1B, a known ID gene. Given our findings it is plausible that loss of function of both genes contributes to the individual's phenotype. This case highlights the complexity of the interpretations of genetic findings from genome-wide investigations. We also performed proteomics analysis of neurons from both the wild-type and Usp9x knockout embryos and identified disruption of the cytoskeleton as the main underlying consequence of the loss of Usp9x. Detailed clinical assessment of all three families with USP9X variants identified hypotonia and behavioral and morphological defects as common features in addition to ID. Together our data support involvement of all three USP9X variants in ID in these families and provide likely cellular and molecular mechanisms involved.

La FAM fatale: USP9X in development and disease.

Deubiquitylating enzymes (DUBs), act downstream of ubiquitylation. As such, these post-post-translational modifiers function as the final arbitrators of a protein substrate's ubiquitylation status, thus regulating its fate. In most instances, DUBs moderate the absolute level of a substrate, its locality or activity, rather than being an "all-or-none" phenomenon. Yet, disruption of this quantitative regulation can produce dramatic qualitative differences. The ubiquitin-specific protease 9X (USP9X/FAM) is a substrate-specific DUB, which displays an extraordinarily high level of sequence conservation from Drosophila to mammals. It is primarily the recent revelations of USP9X's pivotal role in human cancers, both as oncogene or tumour suppressor, in developmental disorders including intellectual disability, epilepsy, autism and developmental delay that has led to a subsequent re-examination of its molecular and cellular functions. Results from experimental animal models have implicated USP9X in neurodegeneration, including Parkinson's and Alzheimer's disease, as well as autoimmune diseases. In this review, we describe the current and accumulated knowledge on the molecular, cellular and developmental aspects of USP9X function within the context of the biological consequences during normal development and disease.

Loss of FMR2 further emphasizes the link between deregulation of immediate early response genes FOS and JUN and intellectual disability.

Loss of FMR2 causes Fragile X E (FRAXE) site-associated intellectual disability (ID). FMR2 regulates transcription, promotes alternative splicing with preference for G-quartet structure harbouring exons and is localized to the nuclear speckles. In primary skin fibroblasts from FRAXE patients (n = 8), we found a significant reduction in the number, but a significant increase in the size, of nuclear speckles, when compared with the controls (n = 4). Since nuclear speckles are enriched with factors involved in pre-mRNA processing, we explored the consequence of these defects and the loss of FMR2 on the transcriptome. We performed whole genome expression profiling using total RNA extracted from these cell lines and found 27 genes significantly deregulated by at least 2-fold at P < 0.05 in the patients. Among these genes, FOS was significantly upregulated and was further investigated due to its established role in neuronal cell function. We showed that (i) 30% depletion of Fmr2 in mouse primary cortical neurons led to a 2-fold increase in Fos expression, (ii) overexpression of FMR2 significantly decreased FOS promoter activity in luciferase assays, and (iii) as FOS promoter contains a serum response element, we found that not FOS, but JUN, which encodes for a protein that forms a transcriptional activator complex with FOS, was significantly upregulated in the patients' cell lines upon mitogen stimulation. These results suggest that FMR2 is an upstream regulator of FOS and JUN, and further link deregulation of the immediate early response genes to the pathology of ID- and FRAXE-associated ID in particular.

The UPF3B gene, implicated in intellectual disability, autism, ADHD and childhood onset schizophrenia regulates neural progenitor cell behaviour and neuronal outgrowth.

Loss-of-function mutations in UPF3B result in variable clinical presentations including intellectual disability (ID, syndromic and non-syndromic), autism, childhood onset schizophrenia and attention deficit hyperactivity disorder. UPF3B is a core member of the nonsense-mediated mRNA decay (NMD) pathway that functions to rapidly degrade transcripts with premature termination codons (PTCs). Traditionally identified in thousands of human diseases, PTCs were recently also found to be part of 'normal' genetic variation in human populations. Furthermore, many human transcripts have naturally occurring regulatory features compatible with 'endogenous' PTCs strongly suggesting roles of NMD beyond PTC mRNA control. In this study, we investigated the role of Upf3b and NMD in neural cells. We provide evidence that suggests Upf3b-dependent NMD (Upf3b-NMD) is regulated at multiple levels during development including regulation of expression and sub-cellular localization of Upf3b. Furthermore, complementary expression of Upf3b, Upf3a and Stau1 stratify the developing dorsal telencephalon, suggesting that alternative NMD, and the related Staufen1-mediated mRNA decay (SMD) pathways are differentially employed. A loss of Upf3b-NMD in neural progenitor cells (NPCs) resulted in the expansion of cell numbers at the expense of their differentiation. In primary hippocampal neurons, loss of Upf3b-NMD resulted in subtle neurite growth effects. Our data suggest that the cellular consequences of loss of Upf3b-NMD can be explained in-part by changes in expression of key NMD-feature containing transcripts, which are commonly deregulated also in patients with UPF3B mutations. Our research identifies novel pathological mechanisms of UPF3B mutations and at least partly explains the clinical phenotype of UPF3B patients.

A noncoding, regulatory mutation implicates HCFC1 in nonsyndromic intellectual disability.

The discovery of mutations causing human disease has so far been biased toward protein-coding regions. Having excluded all annotated coding regions, we performed targeted massively parallel resequencing of the nonrepetitive genomic linkage interval at Xq28 of family MRX3. We identified in the binding site of transcription factor YY1 a regulatory mutation that leads to overexpression of the chromatin-associated transcriptional regulator HCFC1. When tested on embryonic murine neural stem cells and embryonic hippocampal neurons, HCFC1 overexpression led to a significant increase of the production of astrocytes and a considerable reduction in neurite growth. Two other nonsynonymous, potentially deleterious changes have been identified by X-exome sequencing in individuals with intellectual disability, implicating HCFC1 in normal brain function.

Compromised transcription-mRNA export factor THOC2 causes R-loop accumulation, DNA damage and adverse neurodevelopment.

We implicated the X-chromosome THOC2 gene, which encodes the largest subunit of the highly-conserved TREX (Transcription-Export) complex, in a clinically complex neurodevelopmental disorder with intellectual disability as the core phenotype. To study the molecular pathology of this essential eukaryotic gene, we generated a mouse model based on a hypomorphic Thoc2 exon 37-38 deletion variant of a patient with ID, speech delay, hypotonia, and microcephaly. The Thoc2 exon 37-38 deletion male (Thoc2Δ/Y) mice recapitulate the core phenotypes of THOC2 syndrome including smaller size and weight, and significant deficits in spatial learning, working memory and sensorimotor functions. The Thoc2Δ/Y mouse brain development is significantly impacted by compromised THOC2/TREX function resulting in R-loop accumulation, DNA damage and consequent cell death. Overall, we suggest that perturbed R-loop homeostasis, in stem cells and/or differentiated cells in mice and the patient, and DNA damage-associated functional alterations are at the root of THOC2 syndrome.