Cheryl Shoubridge

Constraint and conservation of paired-type homeodomains predicts the clinical outcome of missense variants of uncertain significance.

Publisher: Hindawi Limited

Date: 02-06-2020

DOI: 10.1002/HUMU.24034

Splice variant in ARX leading to loss of C‐terminal region in a boy with intellectual disability and infantile onset developmental and epileptic encephalopathy

Publisher: Wiley

Date: 30-05-2019

DOI: 10.1002/AJMG.A.61216

Abstract: Pathogenic variants in the X-chromosome Aristaless-related homeobox (ARX) gene contribute to intellectual disability, epilepsy, and associated comorbidities in affected males. Here, we report a novel splice variant in ARX in a family with three affected in iduals. The proband had early onset developmental and epileptic encephalopathy, his brother and mother had severe and mild intellectual disability, respectively. Massively parallel sequencing identified a novel c.1449-1G>C in intron 4 of the ARX gene, predicted to abolish the splice acceptor site, retaining intron 4 and leading to a premature termination codon immediately after exon 4. As exon 5 is the last exon of the ARX gene, the premature termination codon at position p.L484* would be predicted to escape nonsense-mediated mRNA decay, potentially producing at least some C-terminally truncated protein. Analysis of cDNA from patient lymphoblastoid cells confirmed retention of intron 4 and loss of detectable expression of ARX mRNA across exon 4 to exon 5. We review published cases of variants that lead to altered or early termination of the ARX protein, but not complete loss of function, and are associated with phenotypes of intellectual disability and infantile onset developmental and epileptic encephalopathies, including Ohtahara and West syndromes. Taken together, this novel splice variant retaining intron 4 is likely to be the cause of the early onset developmental and epileptic encephalopathy in the proband.

Reinitiation of mRNA translation in a patient with X-linked infantile spasms with a protein-truncating variant in ARX

Publisher: Springer Science and Business Media LLC

Date: 26-08-2015

DOI: 10.1038/EJHG.2015.176

ARX polyalanine expansion mutations lead to migration impediment in the rostral cortex coupled with a developmental deficit of calbindin-positive cortical GABAergic interneurons

Publisher: Elsevier BV

Date: 08-2017

DOI: 10.1016/J.NEUROSCIENCE.2017.06.010

Abstract: The Aristaless-related homeobox gene (ARX) is indispensable for interneuron development. Patients with ARX polyalanine expansion mutations of the first two tracts (namely PA1 and PA2) suffer from intellectual disability of varying severity, with seizures a frequent comorbidity. The impact of PA1 and PA2 mutations on the brain development is unknown, hindering the search for therapeutic interventions. Here, we characterized the disturbances to cortical interneuron development in mice modeling the two most common ARX polyalanine expansion mutations in human. We found a consistent ∼40-50% reduction of calbindin-positive interneurons, but not Stt+ or Cr+ interneurons, within the cortex of newborn hemizygous mice (p=0.024) for both mutant strains compared to wildtype (p=0.011). We demonstrate that this was a consequence of calbindin precursor cells being arrested or delayed at the ventral subpallium en route of tangential migration. Ex-vivo assay validated this migration deficit in PA1 cells (p=0.0002) suggesting that the defect is contributed by intrinsic loss of Arx function within migrating cells. Both humans and mice with PA1 mutations present with severe clinical features, including intellectual disability and infantile spasms. Our data further demonstrated the pathogenic mechanism was robustly shared between PA1 and PA2 mutations, as previously reported including Arx protein reduction and overlapping transcriptome profiles within the developing mouse brains. Data from our study demonstrated that cortical calbindin interneuron development and migration is negatively affected by ARX polyalanine expansion mutations. Understanding the cellular pathogenesis contributing to disease manifestation is necessary to screen efficacy of potential therapeutic interventions.

Copy number variants in patients with intellectual disability affect the regulation of ARX transcription factor gene

Publisher: Springer Science and Business Media LLC

Date: 04-09-2015

DOI: 10.1007/S00439-015-1594-X

Abstract: Protein-coding mutations in the transcription factor-encoding gene ARX cause various forms of intellectual disability (ID) and epilepsy. In contrast, variations in surrounding non-coding sequences are correlated with milder forms of non-syndromic ID and autism and had suggested the importance of ARX gene regulation in the etiology of these disorders. We compile data on several novel and some already identified patients with or without ID that carry duplications of ARX genomic region and consider likely genetic mechanisms underlying the neurodevelopmental defects. We establish the long-range regulatory domain of ARX and identify its brain region-specific autoregulation. We conclude that neurodevelopmental disturbances in the patients may not simply arise from increased dosage due to ARX duplication. This is further exemplified by a small duplication involving a non-functional ARX copy, but with duplicated enhancers. ARX enhancers are located within a 504-kb region and regulate expression specifically in the forebrain in developing and adult zebrafish. Transgenic enhancer-reporter lines were used as in vivo tools to delineate a brain region-specific negative and positive autoregulation of ARX. We find autorepression of ARX in the telencephalon and autoactivation in the ventral thalamus. Fluorescently labeled brain regions in the transgenic lines facilitated the identification of neuronal outgrowth and pathfinding disturbances in the ventral thalamus and telencephalon that occur when arxa dosage is diminished. In summary, we have established a model for how breakpoints in long-range gene regulation alter the expression levels of a target gene brain region-specifically, and how this can cause subtle neuronal phenotypes relating to the etiology of associated neuropsychiatric disease.

Embryonic forebrain transcriptome of mice with polyalanine expansion mutations in theARXhomeobox gene

Publisher: Oxford University Press (OUP)

Date: 25-10-2016

DOI: 10.1093/HMG/DDW360

Abstract: The Aristaless-related homeobox (ARX) gene encodes a paired-type homeodomain transcription factor with critical roles in embryonic development. Mutations in ARX give rise to intellectual disability (ID), epilepsy and brain malformation syndromes. To capture the genetics and molecular disruptions that underpin the ARX-associated clinical phenotypes, we undertook a transcriptome wide RNASeq approach to analyse developing (12.5 dpc) telencephalon of mice modelling two recurrent polyalanine expansion mutations with different phenotypic severities in the ARX gene. Here we report 238 genes significantly deregulated (Log2FC > +/-1.1, P-value <0.05) when both mutations are compared to wild-type (WT) animals. When each mutation is considered separately, a greater number of genes were deregulated in the severe PA1 mice (825) than in the PA2 animals (78). Analysing genes deregulated in either or both mutant strains, we identified 12% as implicated in ID, epilepsy and autism (99/858), with ∼5% of them as putative or known direct targets of ARX transcriptional regulation. We propose a core pathway of transcription regulators, including Hdac4, involved in chromatin condensation and transcriptional repression, and one of its targets, the transcription factor Twist1, as potential drivers of the ID and infantile spasms in patients with ARX polyalanine expansion mutations. We predict that the subsequent disturbance to this pathway is a consequence of ARX protein reduction with a broader and more significant level of disruption in the PA1 in comparison to the PA2 mice. Identifying early triggers of ARX-associated phenotypes contributes to our understanding of particular clusters athways underpinning comorbid phenotypes that are shared by many neurodevelopmental disorders.

Polyalanine expansions drive a shift into α-helical clusters without amyloid-fibril formation

Publisher: Springer Science and Business Media LLC

Date: 16-11-2015

DOI: 10.1038/NSMB.3127

Abstract: Polyglutamine (polyGln) expansions in nine human proteins result in neurological diseases and induce the proteins' tendency to form β-rich amyloid fibrils and intracellular deposits. Less well known are at least nine other human diseases caused by polyalanine (polyAla)-expansion mutations in different proteins. The mechanisms of how polyAla aggregates under physiological conditions remain unclear and controversial. We show here that aggregation of polyAla is mechanistically dissimilar to that of polyGln and hence does not exhibit amyloid kinetics. PolyAla assembled spontaneously into α-helical clusters with erse oligomeric states. Such clustering was pervasive in cells irrespective of visible aggregate formation, and it disrupted the normal physiological oligomeric state of two human proteins natively containing polyAla: ARX and SOX3. This self-assembly pattern indicates that polyAla expansions chronically disrupt protein behavior by imposing a deranged oligomeric status.

ARX spectrum disorders: Making inroads into the molecular pathology

Publisher: Hindawi Limited

Date: 17-05-2010

DOI: 10.1002/HUMU.21288

Abstract: The Aristaless-related homeobox gene (ARX) is one of the most frequently mutated genes in a spectrum of X-chromosome phenotypes with intellectual disability (ID) as their cardinal feature. To date, close to 100 families and isolated cases have been reported to carry 44 different mutations, the majority of these (59%) being a result of polyalanine tract expansions. At least 10 well-defined clinical entities, including Ohtahara, Partington, and Proud syndromes, X-linked infantile spasms, X-linked lissencephaly with ambiguous genitalia, X-linked myoclonic epilepsy and nonsyndromic intellectual disability have been ascertained from among the patients with ARX mutations. The striking intra- and interfamilial pleiotropy together with genetic heterogeneity (same clinical entities associated with different ARX mutations) are becoming a hallmark of ARX mutations. Although males are predominantly affected, some mutations associated with malformation phenotypes in males also show a phenotype in carrier females. Recent progress in the study of the effect of ARX mutations through sophisticated animal (mice) and cellular models begins to provide crucial insights into the molecular function of ARX and associated molecular pathology, thus guiding future inquiries into therapeutic interventions.

Polyalanine tract disorders and neurocognitive phenotypes

Publisher: Springer New York

Date: 2012

DOI: 10.1007/978-1-4614-5434-2_12

Abstract: Expansion of polyalanine tracts cause at least 9 inherited human diseases. Eight of these nine diseases are due to expansions in transcription factors and give rise to congenital disorders, many with neurocognitive phenotypes. Disease-causing expansions vary in length dependingupon the gene in question, with the severity of the associated clinical phenotype generally increasing with length of the polyalanine tract. The past decade has seen considerable progress in the understanding on how these mutations may arise and the functional effect of expanded polyalanine tracts on the resulting protein. Despite this progress, the pathogenic mechanism of expanded polyalanine tracts contributing to the associated disease states remains poorly understood. Gaining insights into the mechanisms that underlie the pathogenesis of different expanded polyalanine tract mutations will be a necessary step on the path to the design of potential treatment strategies for the associated diseases.

Histone demethylase KDM5C is a SAHA-sensitive central hub at the crossroads of transcriptional axes involved in multiple neurodevelopmental disorders

Publisher: Oxford University Press (OUP)

Date: 06-11-2019

DOI: 10.1093/HMG/DDZ254

Abstract: A disproportional large number of neurodevelopmental disorders (NDDs) is caused by variants in genes encoding transcription factors and chromatin modifiers. However, the functional interactions between the corresponding proteins are only partly known. Here, we show that KDM5C, encoding a H3K4 demethylase, is at the intersection of transcriptional axes under the control of three regulatory proteins ARX, ZNF711 and PHF8. Interestingly, mutations in all four genes (KDM5C, ARX, ZNF711 and PHF8) are associated with X-linked NDDs comprising intellectual disability as a core feature. in vitro analysis of the KDM5C promoter revealed that ARX and ZNF711 function as antagonist transcription factors that activate KDM5C expression and compete for the recruitment of PHF8. Functional analysis of mutations in these genes showed a correlation between phenotype severity and the reduction in KDM5C transcriptional activity. The KDM5C decrease was associated with a lack of repression of downstream target genes Scn2a, Syn1 and Bdnf in the embryonic brain of Arx-null mice. Aiming to correct the faulty expression of KDM5C, we studied the effect of the FDA-approved histone deacetylase inhibitor suberanilohydroxamic acid (SAHA). In Arx-KO murine ES-derived neurons, SAHA was able to rescue KDM5C depletion, recover H3K4me3 signalling and improve neuronal differentiation. Indeed, in ARX/alr-1-deficient Caenorhabditis elegans animals, SAHA was shown to counteract the defective KDM5C/rbr-2-H3K4me3 signalling, recover abnormal behavioural phenotype and ameliorate neuronal maturation. Overall, our studies indicate that KDM5C is a conserved and druggable effector molecule across a number of NDDs for whom the use of SAHA may be considered a potential therapeutic strategy.

Incorrect dosage of IQSEC2, a known intellectual disability and epilepsy gene, disrupts dendritic spine morphogenesis.

Publisher: Springer Science and Business Media LLC

Date: 02-05-2017

DOI: 10.1038/TP.2017.81

Abstract: There is considerable genetic and phenotypic heterogeneity associated with intellectual disability (ID), specific learning disabilities, attention-deficit hyperactivity disorder, autism and epilepsy. The intelligence quotient (IQ) motif and SEC7 domain containing protein 2 gene ( IQSEC2 ) is located on the X-chromosome and harbors mutations that contribute to non-syndromic ID with and without early-onset seizure phenotypes in both sexes. Although IQ and Sec7 domain mutations lead to partial loss of IQSEC2 enzymatic activity, the in vivo pathogenesis resulting from these mutations is not known. Here we reveal that IQSEC2 has a key role in dendritic spine morphology. Partial loss-of-function mutations were modeled using a lentiviral short hairpin RNA (shRNA) approach, which achieved a 57% knockdown of Iqsec2 expression in primary hippoc al cell cultures from mice. Investigating gross morphological parameters after 8 days of in vitro culture (8DIV) identified a 32% reduction in primary axon length, in contrast to a 27% and 31% increase in the number and complexity of dendrites protruding from the cell body, respectively. This increase in dendritic complexity and spread was carried through dendritic spine development, with a 34% increase in the number of protrusions per dendritic segment compared with controls at 15DIV. Although the number of dendritic spines had normalized by 21DIV, a reduction was noted in the number of immature spines. In contrast, when modeling increased dosage, overexpression of wild-type IQSEC2 led to neurons with shorter axons that were more compact and displayed simpler dendritic branching. Disturbances to dendritic morphology due to knockdown of Iqsec2 were recapitulated in neurons from Iqsec2 knockout mice generated in our laboratory using CRISPR/Cas9 technology. These observations provide evidence of dosage sensitivity for IQSEC2 , which normally escapes X-inactivation in females, and links these disturbances in expression to alterations in the morphology of developing neurons.

Unraveling the pathogenesis of ARX polyalanine tract variants using a clinical and molecular interfacing approach

Publisher: Wiley

Date: 25-02-2015

DOI: 10.1002/MGG3.133

Preferential Intestinal Delivery of Long[Arg³] Insulin-Like Growth Factor (LR³IGF-I) over IGF-I in Preweaning and Adult Rats

Publisher: The Endocrine Society

Date: 05-2003

DOI: 10.1210/EN.2002-220643

Abstract: During early postnatal development, the intestine is highly responsive to LR(3)IGF-I administration but refractory to IGF-I, in contrast to the mature intestine. Given that LR(3)IGF-I is an IGF-I analog that binds poorly to IGF binding proteins, the response of the intestine is likely to reflect regulation of IGF-I bioactivity by IGF binding proteins. This study measures the delivery of exogenous IGF-I peptides to the intestine in preweaning (d-19) and adult rats to determine whether a correlation exists with the potency advantage of LR(3)IGF-I in the intestine during postnatal development. IGF-I or LR(3)IGF-I (2.6 microg/kg) was spiked with corresponding (125)I-labeled peptide (10 x 10(6) cpm) and administered iv as a bolus (n = 5-6/group) with blood and tissue s les collected 5 and 10 min post injection. In both age groups, the levels of (125)I-IGF-I retained in the blood at both 5 and 10 min were higher than the levels of (125)I-LR(3)IGF-I, consistent with the slower clearance rate for the native peptide. In the gastrointestinal tract, the levels of (125)I-LR(3)IGF-I per gram of tissue were 37-50% higher than (125)I-IGF-I. Surprisingly, there was little difference in the relative delivery of LR(3)IGF-I to IGF-I to the intestine, across developmental age. Although bolus iv-injected LR(3)IGF-I was cleared more rapidly from the circulation than IGF-I and was subsequently delivered to the intestine in higher amounts than the native peptide, the ratio of LR(3)IGF-I to IGF-I in gut tissues was approximately 2:1 in both age groups. Hence, selective delivery to the gut is unlikely to explain the markedly higher potency of (125)I-LR(3)IGF-I in stimulating growth of the preweaning vs. adult intestine.

A Regulatory Path Associated with X-Linked Intellectual Disability and Epilepsy Links KDM5C to the Polyalanine Expansions in ARX

Publisher: Elsevier BV

Date: 2013

DOI: 10.1016/J.AJHG.2012.11.008

Diagnostic Yield of Whole Genome Sequencing After Nondiagnostic Exome Sequencing or Gene Panel in Developmental and Epileptic Encephalopathies

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 30-03-2021

DOI: 10.1212/WNL.0000000000011655

Abstract: To assess the benefits and limitations of whole genome sequencing (WGS) compared to exome sequencing (ES) or multigene panel (MGP) in the molecular diagnosis of developmental and epileptic encephalopathies (DEE). We performed WGS of 30 comprehensively phenotyped DEE patient trios that were undiagnosed after first-tier testing, including chromosomal microarray and either research ES (n = 15) or diagnostic MGP (n = 15). Eight diagnoses were made in the 15 in iduals who received prior ES (53%): 3 in iduals had complex structural variants 5 had ES-detectable variants, which now had additional evidence for pathogenicity. Eleven diagnoses were made in the 15 MGP-negative in iduals (68%) the majority (n = 10) involved genes not included in the panel, particularly in in iduals with postneonatal onset of seizures and those with more complex presentations including movement disorders, dysmorphic features, or multiorgan involvement. A total of 42% of diagnoses were autosomal recessive or X-chromosome linked. WGS was able to improve diagnostic yield over ES primarily through the detection of complex structural variants (n = 3). The higher diagnostic yield was otherwise better attributed to the power of re-analysis rather than inherent advantages of the WGS platform. Additional research is required to assist in the assessment of pathogenicity of novel noncoding and complex structural variants and further improve diagnostic yield for patients with DEE and other neurogenetic disorders.

A novel ARX loss of function variant in female monozygotic twins is associated with chorea

Publisher: Elsevier BV

Date: 11-2021

DOI: 10.1016/J.EJMG.2021.104315

Abstract: Pathogenic variants in ARX lead to a variety of phenotypes with intellectual disability being a uniform feature. Other features can include severe epilepsy, spasticity, movement disorders, agenesis of the corpus callosum, lissencephaly, hydranencephaly and ambiguous genitalia in males. We present the first report of monozygotic female twins with a de novo ARX pathogenic variant (c.1406_1415del p. Ala469Aspfs*20), predicted to result in a truncated ARX protein missing the important regulatory Aristaless domain. The twins presented with profound developmental delay and seizures, consistent with the known genotype-phenotype correlation. Twin 2's features were significantly more severe. She also developed chorea the first time this movement disorder has been seen in an ARX variant other than an expansion of the first polyalanine tract. Differential X-chromosome inactivation was the most likely explanation for the differing severities but could not be conclusively proven.

Heterozygous loss of function of IQSEC2/Iqsec2 leads to increased activated Arf6 and severe neurocognitive seizure phenotype in females

Publisher: Life Science Alliance, LLC

Date: 08-2019

DOI: 10.26508/LSA.201900386

Abstract: Clinical presentations of mutations in the IQSEC2 gene on the X-chromosome initially implicated to cause non-syndromic intellectual disability (ID) in males have expanded to include early onset seizures in males as well as in females. The molecular pathogenesis is not well understood, nor the mechanisms driving disease expression in heterozygous females. Using a CRISPR/Cas9–edited Iqsec2 KO mouse model, we confirm the loss of Iqsec2 mRNA expression and lack of Iqsec2 protein within the brain of both founder and progeny mice. Both male (52%) and female (46%) Iqsec2 KO mice present with frequent and recurrent seizures. Focusing on Iqsec2 KO heterozygous female mice, we demonstrate increased hyperactivity, altered anxiety and fear responses, decreased social interactions, delayed learning capacity and decreased memory retention/novel recognition, recapitulating psychiatric issues, autistic-like features, and cognitive deficits present in female patients with loss-of-function IQSEC2 variants. Despite Iqsec2 normally acting to activate Arf6 substrate, we demonstrate that mice modelling the loss of Iqsec2 function present with increased levels of activated Arf6. We contend that loss of Iqsec2 function leads to altered regulation of activated Arf6-mediated responses to synaptic signalling and immature synaptic networks. We highlight the importance of IQSEC2 function for females by reporting a novel nonsense variant c.566C A, p.(S189*) in an elderly female patient with profound intellectual disability, generalised seizures, and behavioural disturbances. Our human and mouse data reaffirm IQSEC2 as another disease gene with an unexpected X-chromosome heterozygous female phenotype. Our Iqsec2 mouse model recapitulates the phenotypes observed in human patients despite the differences in the IQSEC2/Iqsec2 gene X-chromosome inactivation between the species.

Mutations in the guanine nucleotide exchange factor gene IQSEC2 cause nonsyndromic intellectual disability

Publisher: Springer Science and Business Media LLC

Date: 16-05-2010

DOI: 10.1038/NG.588

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

Publisher: Elsevier BV

Date: 10-2012

DOI: 10.1016/J.AJHG.2012.08.011

Xp11.2 microduplications including IQSEC2, TSPYL2 and KDM5C genes in patients with neurodevelopmental disorders

Publisher: Springer Science and Business Media LLC

Date: 10-06-2015

DOI: 10.1038/EJHG.2015.123

IQSEC2 mutation update and review of the female-specific phenotype spectrum including intellectual disability and epilepsy.

Publisher: Hindawi Limited

Date: 08-11-2018

DOI: 10.1002/HUMU.23670

Abstract: The IQSEC2- related disorders represent a spectrum of X-chromosome phenotypes with intellectual disability (ID) as the cardinal feature. Here, we review the increasing number of reported families and isolated cases have been reported with a variety of different pathogenic variants. The spectrum of clinical features is expanding with early-onset seizures as a frequent comorbidity in both affected male and female patients. There is a growing number of female patients with de novo loss-of-function variants in IQSEC2 have a more severe phenotype than the heterozygous state would predict, particularly if IQSEC2 is thought to escape X-inactivation. Interestingly, these findings highlight that the classical understanding of X-linked inheritance does not readily explain the emergence of these affected females, warranting further investigations into the underlying mechanisms.

Enhancement of intestinal growth and repair by growth factors

Publisher: Elsevier BV

Date: 12-2001

DOI: 10.1016/S1471-4892(01)00098-4

Abstract: Recently, glucagon-like peptide 2 has emerged as a potent stimulator of epithelial growth, joining insulin-like growth factor I, hepatocyte growth factor and keratinocyte growth factor as potential treatment modalities for intestinal disorders associated with loss of mucosal mass, such as short bowel syndrome. Investigations into other members of the expanded epidermal growth factor peptide family, the development of more potent peptide analogues, and advances in the development of enterally administered bioactive growth factor formulations further expands the repertoire of epithelial growth factors applicable to conditions associated with epithelial insufficiency.

Extensive phenotyping of two ARX polyalanine expansion mutation mouse models that span clinical spectrum of intellectual disability and epilepsy

Publisher: Elsevier BV

Date: 09-2017

DOI: 10.1016/J.NBD.2017.05.012

Abstract: The Aristaless-related homeobox gene (ARX) is a known intellectual disability (ID) gene that frequently presents with X-linked infantile spasm syndrome as a comorbidity. ID with epilepsy in children is a chronic and devastating disorder that has poor treatment options and disease outcomes. To gain a better understanding of the role that mutations in ARX play in ID and epilepsy, we investigate ARX patient mutations modelled in mice. Over half of all ARX mutations result from expansions of the first two polyalanine (PA1 and PA2 respectively) tracts. However, phenotypic data for the mouse modelling the more frequent ARX PA2 dup24 mutation in patients has not been reported and constitutes a barrier to understanding the molecular mechanisms involved. Here we report the first comprehensive analysis of postnatal outcomes for mice modelling disease-causing expansions to both PA1 and PA2 tracts. Both strains were found to have impaired learning and memory, reduced activity, increased anxiety and reduced sociability with PA1 mice generally displaying greater behavioural deficits in keeping with the more severe phenotype reported in patients. In agreement with previous reports, 70% of PA1 males exhibit myoclonic seizures by two months of age, with the first observed at P18. In this report, we show 80% of PA2 males also display myoclonic seizures, with the first observed at P19. Consistent with patient phenotypes, we observe large variations in seizure progression and severity for both PA1 and PA2 in idual mice. The generation of this comprehensive baseline data is a necessary step on the path to the development of therapies to improve patient outcomes.

Molecular pathology of expanded polyalanine tract mutations in the Aristaless-related homeobox gene

Publisher: Elsevier BV

Date: 07-2007

DOI: 10.1016/J.YGENO.2007.03.005

Abstract: The Aristaless-related homeobox gene (ARX) is one of the major genes causing X-linked mental retardation. We have been interested in the pathogenic mechanism of expanded polyalanine tract mutations in ARX. We showed that the c.304ins(GCG)7 mutation causing an increase from 16 to 23 alanines increased the propensity of ARX protein aggregation and a shift from nuclear to cytoplasmic localization. We proposed that mislocalization of ARX via cytoplasmic aggregation and subsequent degradation leads to a partial loss of function, contributing to the pathogenesis. We identified importin 13 (IPO13), a mediator of nuclear import for a variety of proteins, as a novel ARX interacting protein. We predicted that the transport of ARX by IPO13 from the cytoplasm to the nucleus might be disrupted by expanded polyalanine tract mutations, but our data showed that in both yeast and mammalian cells these mutant ARX proteins were still able to interact with IPO13. We established the nuclear localization regions of the ARX homeodomain that were required for the interaction with IPO13 and correct localization of the full-length ARX transcription factor to the nucleus.

A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation

Publisher: Springer Science and Business Media LLC

Date: 19-04-2009

DOI: 10.1038/NG.367

Early 17β-estradiol treatment reduces seizures but not abnormal behaviour in mice with expanded polyalanine tracts in the Aristaless related homeobox gene (ARX)

Publisher: Elsevier BV

Date: 06-2021

DOI: 10.1016/J.NBD.2021.105329

Abstract: Children with severe intellectual disability have an increased prevalence of refractory seizures. Steroid treatment may improve seizure outcomes, but the mechanism remains unknown. Here we demonstrate that short term, daily delivery of an exogenous steroid 17β-estradiol (40 ng/g) in early postnatal life significantly reduced the number and severity of seizures, but did not improve behavioural deficits, in mice modelling mutations in the Aristaless-related homeobox gene (ARX), expanding the first (PA1) or second (PA2) polyalanine tract. Frequency of observed seizures on handling (n = 14/treatment/genotype) were significantly reduced in PA1 (32% reduction) and more modestly reduced in PA2 mice (14% reduction) with steroid treatment compared to vehicle. Spontaneous seizures were assessed (n = 7/treatment/genotype) at 7 weeks of age coinciding with a peak of seizure activity in untreated mice. PA1 mice treated with steroids no longer present with the most severe category of prolonged myoclonic seizures. Treated PA2 mice had an earlier onset of seizures coupled with a subsequent reduction in seizures later in postnatal life, with a complete absence of any seizures during the analysis at 7 weeks of age. Despite the reduction in seizures, 17β-estradiol treated mice showed no improvement in behavioural or cognitive outcomes in adulthood. For the first time we show that these deficits due to mutations in Arx are already present before seizure onset and do not worsen with seizures. ARX is a transcription factor and Arx PA mutant mice have deregulated transcriptome profiles in the developing embryonic brain. At postnatal day 10, treatment completion, RNAseq identified 129 genes significantly deregulated (Log2FC > ± 0.5, P-value<0.05) in the frontal cortex of mutant compared to wild-type mice. This list reflects genes deregulated in disease and was particularly enriched for known genes in neurodevelopmental disorders and those involved in signalling and developmental pathways. 17β-estradiol treatment of mutant mice significantly deregulated 295 genes, with only 23 deregulated genes overlapping between vehicle and steroid treated mutant mice. We conclude that 17β-estradiol treatment recruits processes and pathways to reduce the frequency and severity of seizures in the Arx PA mutant mice but does not precisely correct the deregulated transcriptome nor improve mortality or behavioural and cognitive deficits.

Mutations of the UPF3B gene, which encodes a protein widely expressed in neurons, are associated with nonspecific mental retardation with or without autism

Publisher: Springer Science and Business Media LLC

Date: 24-02-2009

DOI: 10.1038/MP.2009.14

Abstract: Mutations in the UPF3B gene, which encodes a protein involved in nonsense-mediated mRNA decay, have recently been described in four families with specific (Lujan-Fryns and FG syndromes), nonspecific X-linked mental retardation (XLMR) and autism. To further elucidate the contribution of UPF3B to mental retardation (MR), we screened its coding sequence in 397 families collected by the EuroMRX consortium. We identified one nonsense mutation, c.1081C>T .Arg361(*), in a family with nonspecific MR (MRX62) and two amino-acid substitutions in two other, unrelated families with MR and/or autism (c.1136G>A .Arg379His and c.1103G>A .Arg368Gln). Functional studies using lymphoblastoid cell lines from affected patients revealed that c.1081C>T mutation resulted in UPF3B mRNA degradation and consequent absence of the UPF3B protein. We also studied the subcellular localization of the wild-type and mutated UPF3B proteins in mouse primary hippoc al neurons. We did not detect any obvious difference in the localization between the wild-type UPF3B and the proteins carrying the two missense changes identified. However, we show that UPF3B is widely expressed in neurons and also presents in dendritic spines, which are essential structures for proper neurotransmission and thus learning and memory processes. Our results demonstrate that in addition to Lujan-Fryns and FG syndromes, UPF3B protein truncation mutations can cause also nonspecific XLMR. We also identify comorbidity of MR and autism in another family with UPF3B mutation. The neuronal localization pattern of the UPF3B protein and its function in mRNA surveillance suggests a potential function in the regulation of the expression and degradation of various mRNAs present at the synapse.

The molecular and phenotypic spectrum of IQSEC2-related epilepsy.

Publisher: Wiley

Date: 26-09-2016

DOI: 10.1111/EPI.13560

Abstract: IQSEC2 is an X-linked gene associated with intellectual disability (ID) and epilepsy. Herein we characterize the epilepsy/epileptic encephalopathy of patients with IQSEC2 pathogenic variants. Forty-eight patients with IQSEC2 variants were identified worldwide through Medline search. Two patients were recruited from our early onset epileptic encephalopathy cohort and one patient from personal communication. The 18 patients who have epilepsy in addition to ID are the subject of this study. Information regarding the 18 patients was ascertained by questionnaire provided to the treating clinicians. Six affected in iduals had an inherited IQSEC2 variant and 12 had a de novo one (male-to-female ratio, 12:6). The pathogenic variant types were as follows: missense (8), nonsense (5), frameshift (1), intragenic duplications (2), translocation (1), and insertion (1). An epileptic encephalopathy was diagnosed in 9 (50%) of 18 patients. Seizure onset ranged from 8 months to 4 years seizure types included spasms, atonic, myoclonic, tonic, absence, focal seizures, and generalized tonic-clonic (GTC) seizures. The electroclinical syndromes could be defined in five patients: late-onset epileptic spasms (three) and Lennox-Gastaut or Lennox-Gastaut-like syndrome (two). Seizures were pharmacoresistant in all affected in iduals with epileptic encephalopathy. The epilepsy in the other nine patients had a variable age at onset from infancy to 18 years seizure types included GTC and absence seizures in the hereditary cases and GTC and focal seizures in de novo cases. Seizures were responsive to medical treatment in most cases. All 18 patients had moderate to profound intellectual disability. Developmental regression, autistic features, hypotonia, strabismus, and white matter changes on brain magnetic resonance imaging (MRI) were prominent features. The phenotypic spectrum of IQSEC2 disorders includes epilepsy and epileptic encephalopathy. Epileptic encephalopathy is a main clinical feature in sporadic cases. IQSEC2 should be evaluated in both male and female patients with an epileptic encephalopathy.

Gonadal mosaicism of a novel IQSEC2 variant causing female limited intellectual disability and epilepsy

Publisher: Springer Science and Business Media LLC

Date: 15-03-2017

DOI: 10.1038/EJHG.2017.29

Mutations in UPF3B, a member of the nonsense-mediated mRNA decay complex, cause syndromic and nonsyndromic mental retardation

Publisher: Springer Science and Business Media LLC

Date: 19-08-2007

DOI: 10.1038/NG2100

Different types of disease-causing noncoding variants revealed by genomic and gene expression analyses in families with X-linked intellectual disability

Publisher: Hindawi Limited

Date: 03-05-2021

DOI: 10.1002/HUMU.24207

Abstract: The pioneering discovery research of X-linked intellectual disability (XLID) genes has benefitted thousands of in iduals worldwide however, approximately 30% of XLID families still remain unresolved. We postulated that noncoding variants that affect gene regulation or splicing may account for the lack of a genetic diagnosis in some cases. Detecting pathogenic, gene-regulatory variants with the same sensitivity and specificity as structural and coding variants is a major challenge for Mendelian disorders. Here, we describe three pedigrees with suggestive XLID where distinctive phenotypes associated with known genes guided the identification of three different noncoding variants. We used comprehensive structural, single-nucleotide, and repeat expansion analyses of genome sequencing. RNA-Seq from patient-derived cell lines, reverse-transcription polymerase chain reactions, Western blots, and reporter gene assays were used to confirm the functional effect of three fundamentally different classes of pathogenic noncoding variants: a retrotransposon insertion, a novel intronic splice donor, and a canonical splice variant of an untranslated exon. In one family, we excluded a rare coding variant in ARX, a known XLID gene, in favor of a regulatory noncoding variant in OFD1 that correlated with the clinical phenotype. Our results underscore the value of genomic research on unresolved XLID families to aid novel, pathogenic noncoding variant discovery.

Placental transcriptome co-expression analysis reveals conserved regulatory programs across gestation.

Publisher: Springer Science and Business Media LLC

Date: 03-01-2017

DOI: 10.1186/S12864-016-3384-9

IQSEC2-related encephalopathy in males due to missense variants in the pleckstrin homology domain

Publisher: Wiley

Date: 06-04-2022

DOI: 10.1111/CGE.14136

Abstract: Pathogenic variants in IQ motif and SEC7 domain containing protein 2 ( IQSEC2 ) gene cause a variety of neurodevelopmental disorders, with intellectual disability as a uniform feature. We report five cases, each with a novel missense variant in the pleckstrin homology (PH) domain of the IQSEC2 protein. Male patients all present with moderate to profound intellectual disability, significant delays or absent language and speech and variable seizures. We describe the phenotypic spectrum associated with missense variants in PH domain of IQSEC2 , further delineating the genotype–phenotype correlation for this X‐linked gene.

X-Linked Lissencephaly With Absent Corpus Callosum and Abnormal Genitalia

Publisher: SAGE Publications

Date: 2017

DOI: 10.1177/2329048X17738625

Abstract: X-linked lissencephaly with abnormal genitalia is a rare and devastating syndrome. The authors present an infant with a multisystem phenotype where the intestinal manifestations were as life limiting as the central nervous system features. Severe chronic diarrhea resulted in failure to thrive, dehydration, electrolyte derangements, long-term hospitalization, and prompted transition to palliative care. Other multisystem manifestations included megacolon, colitis, pancreatic insufficiency hypothalamic dysfunction, hypothyroidism, and hypophosphatasia. A novel aristaless-related homeobox gene mutation, c.1136G T .R379L, was identified. This case contributes to the clinical, histological, and molecular understanding of the multisystem nature of this disorder, especially the role of ARX in the development of the enteroendocrine system.

Subtle functional defects in the Arf-specific guanine nucleotide exchange factor IQSEC2 cause non-syndromic X-linked intellectual disability

Publisher: Informa UK Limited

Date: 09-2010

DOI: 10.4161/SGTP.1.2.13285

Mutations of protocadherin 19 in female epilepsy (PCDH19-FE) lead to allopregnanolone deficiency.

Publisher: Oxford University Press (OUP)

Date: 29-06-2015

DOI: 10.1093/HMG/DDV245

Abstract: Protocadherin 19 (PCDH19) female limited epilepsy (PCDH19-FE also known as epilepsy and mental retardation limited to females, EFMR MIM300088) is an infantile onset epilepsy syndrome with or without intellectual disability (ID) and autism. We investigated transcriptomes of PCDH19-FE female and control primary skin fibroblasts, which are endowed to metabolize neurosteroid hormones. We identified a set of 94 significantly dysregulated genes in PCDH19-FE females. Intriguingly, 43 of the 94 genes (45.7%) showed gender-biased expression enrichment of such genes was highly significant (P = 2.51E-47, two-tailed Fisher exact test). We further investigated the AKR1C1-3 genes, which encode crucial steroid hormone-metabolizing enzymes whose key products include allopregnanolone and estradiol. Both mRNA and protein levels of AKR1C3 were significantly decreased in PCDH19-FE patients. In agreement with this, the blood levels of allopregnanolone were also (P < 0.01) reduced. In conclusion, we show that the deficiency of neurosteroid allopregnanolone, one of the most potent GABA receptor modulators, may contribute to PCDH19-FE. Overall our findings provide evidence for a role of neurosteroids in epilepsy, ID and autism and create realistic opportunities for targeted therapeutic interventions.

Is there a Mendelian transmission ratio distortion of the c.429 -452dup(24bp) polyalanine tract ARX mutation?

Publisher: Springer Science and Business Media LLC

Date: 11-04-2012

DOI: 10.1038/EJHG.2012.61

Screening and cell-based assessment of mutations in the Aristaless-related homeobox (ARX) gene

Publisher: Wiley

Date: 18-05-2011

DOI: 10.1111/J.1399-0004.2011.01685.X

Abstract: ARX mutations cause a erse spectrum of human disorders, ranging from severe brain and genital malformations to non-syndromic intellectual disability (ID). ARX is a transcription factor with multiple domains that include four polyalanine (pA) tracts, the first two of which are frequently expanded by mutations. We progressively screened DNA s les from 613 in iduals with ID initially for the most frequent ARX mutations (c.304ins(GCG)(7)'expansion' of pA1 and c.429_452dup 'dup24bp' of pA2). Five hundred s les without pA1 or pA2 mutations had the entire ARX ORF screened by single stranded polymorphism conformation (SSCP) and/or denaturing high pressure liquid chromatography (dHPLC) analysis. Overall, eight families with six mutations in ARX were identified (1.31%): five duplication mutations in pA2 (0.82%) with three new clinical reports of families with the dup24bp and two duplications larger than the dup24bp mutation discovered (dup27bp, dup33bp) and three point mutations (0.6%), including one novel mutation in the homeodomain (c.1074G>T). Four ultraconserved regions distal to ARX (uc466-469) were also screened in a subset of 94 patients, with three unique nucleotide changes identified in two (uc466, uc467). The subcellular localization of full length ARX proteins was assessed for 11 variants. Protein mislocalization increased as a function of pA2 tract length and phenotypic severity, as has been previously suggested for pA1. Similarly, protein mislocalization of the homeodomain mutations also correlated with clinical severity, suggesting an emerging genotype vs cellular phenotype correlation.

Regulating transcriptional activity by phosphorylation: A new mechanism for the ARX homeodomain transcription factor

Publisher: Public Library of Science (PLoS)

Date: 12-11-2018

DOI: 10.1371/JOURNAL.PONE.0206914

An Emerging Female Phenotype with Loss-of-Function Mutations in the Aristaless-Related Homeodomain Transcription Factor ARX.

Publisher: Hindawi Limited

Date: 15-02-2017

DOI: 10.1002/HUMU.23190

Abstract: The devastating clinical presentation of X-linked lissencephaly with abnormal genitalia (XLAG) is invariably caused by loss-of-function mutations in the Aristaless-related homeobox (ARX) gene. Mutations in this X-chromosome gene contribute to intellectual disability (ID) with co-morbidities including seizures and movement disorders such as dystonia in affected males. The detection of affected females with mutations in ARX is increasing. We present a family with multiple affected in iduals, including two females. Two male siblings presenting with XLAG were deceased prior to full-term gestation or within the first few weeks of life. Of the two female siblings, one presented with behavioral disturbances, mild ID, a seizure disorder, and complete agenesis of the corpus callosum (ACC), similar to the mother's phenotype. A novel insertion mutation in Exon 2 of ARX was identified, c.982delCinsTTT predicted to cause a frameshift at p.(Q328Ffs

ARX homeodomain mutations abolish DNA binding and lead to a loss of transcriptional repression

Publisher: Oxford University Press (OUP)

Date: 21-12-2012

DOI: 10.1093/HMG/DDR601

Abstract: Mutations in the Aristaless-related homeobox (ARX) gene are one of the most frequent causes of X-linked intellectual disability (ID). Several missense mutations, clustered in the paired-type homeodomain of ARX, have been identified. These mutations lead to a range of phenotypes from X-linked lissencephaly with abnormal genitalia to seizure disorders without brain malformations including X-linked infantile spasms with ID (ISSX-ID) and X-linked myoclonic epilepsy with spasticity and ID (XMESID). The effect of these mutations on the DNA-binding and transcriptional activity has been evaluated. Luciferase reporter assays showed altered repression activity of ARX by all mutations, causing brain malformations and ISSX-ID phenotypes, but not by the P353L mutation implicated in a milder phenotype of XMESID. Similarly, transient overexpression of wild-type ARX repressed endogenous expression of known ARX targets, LMO1 and SHOX2, when measured by real-time quantitative polymerase chain reaction. Overall, the molecular consequence of missense mutations correlated well with the severity of the clinical phenotype. In all mutations tested, except P353L, the DNA binding was abolished. Electrophoretic mobility shift assay results were validated using chromatin immunoprecipitation following overexpression of normal and selected missense mutations. Unlike wild-type ARX and clinically less severe mutations, the mutations leading to severe clinical phenotypes were not able to specifically bind to DNA upstream of known, endogenous ARX-regulated genes, LMO1 and SHOX2. In conclusion, the missense mutations in the ARX homeodomain represent loss-of-function mutations, which lead to a reduced or complete loss of DNA binding and as a consequence, a loss of transcriptional repression.

The genetic landscape of intellectual disability arising from chromosome X

Publisher: Elsevier BV

Date: 07-2009

DOI: 10.1016/J.TIG.2009.05.002

Abstract: X-linked mental retardation (XLMR) or intellectual disability (ID) is a common, clinically complex and genetically heterogeneous disease arising from many mutations along the X chromosome. It affects between 1/600-1/1000 males and a substantial number of females. Research during the past decade has identified >90 different XLMR genes, affecting a wide range of cellular processes. Many more genes remain uncharacterized, especially for the non-syndromic XLMR forms. Currently, approximately 11% of X-chromosome genes are implicated in XLMR however, apart from a few notable exceptions, most contribute in idually to <0.1% of the total landscape, which arguably remains only about half complete. There remain many hills to climb and valleys to cross before the ID landscape is fully triangulated.

Mutations in the nuclear localization sequence of the Aristaless related homeobox; sequestration of mutant ARX with IPO13 disrupts normal subcellular distribution of the transcription factor and retar

Publisher: Springer Science and Business Media LLC

Date: 05-01-2010

DOI: 10.1186/1755-8417-3-1

Reduced polyalanine-expanded arx mutant protein in developing mouse subpallium alters lmo1 transcriptional regulation

Publisher: Oxford University Press (OUP)

Date: 10-10-2014

DOI: 10.1093/HMG/DDT503

Abstract: Intellectual disability (ID) is a highly prevalent disorder that affects 1-3% of the population. The Aristaless-related homeobox gene (ARX) is a frequently mutated X-linked ID gene and encodes a transcription factor indispensable for proper forebrain, testis and pancreas development. Polyalanine expansions account for over half of all mutations in ARX and clinically give rise to a spectrum of ID and seizures. To understand how the polyalanine expansions cause the clinical phenotype, we studied mouse models of the two most frequent polyalanine expansion mutations (Arx((GCG)7) and Arx(432-455dup24)). Neither model showed evidence of protein aggregates however, a marked reduction of Arx protein abundance within the developing forebrain was striking. Examining the expression of known Arx target genes, we found a more prominent loss of Lmo1 repression in Arx((GCG7)/Y) compared with Arx(432-455dup24/Y) mice at 12.5 and 14.5 dpc, stages of peak neural proliferation and neurogenesis, respectively. Once neurogenesis concludes both mutant mouse models showed similar loss of Lmo1 repression. We propose that this temporal difference in the loss of Lmo1 repression may be one of the causes accounting for the phenotypic differences identified between the Arx((GCG)7)and Arx(432-455dup24) mouse models. It is yet to be determined what effect these mutations have on ARX protein in affected males in the human setting.

Protein and gene expression analysis of Phf6, the gene mutated in the Börjeson-Forssman-Lehmann Syndrome of intellectual disability and obesity

Publisher: Elsevier BV

Date: 10-2007

DOI: 10.1016/J.MODGEP.2007.06.007

Abstract: The Plant homeodomain finger gene 6 (PHF6) was identified as the gene mutated in patients suffering from the Börjeson-Forssman-Lehmann Syndrome (BFLS), an X-linked mental retardation disorder. BFLS mental disability is evident from an early age, suggesting a developmental brain defect. The PHF6 protein contains four nuclear localisation signals and two imperfect plant homeodomain (PHD) fingers similar to the third, imperfect PHD fingers in members of the trithorax family of transcriptional regulators. The PHF6 gene is highly conserved in vertebrate species. Despite the devastating effects of mutation of the PHF6 gene, nothing is known about the cellular function of PHF6. In order to lay the base for functional studies, we identify here the cell types that express the murine Phf6 gene and protein during prenatal and postnatal development. The Phf6 gene and protein are expressed widely. However, expression levels vary from strong to barely detectable. Strongest Phf6 gene expression and nuclear localisation of Phf6 protein were observed in the developing central nervous system, the anterior pituitary gland, the primordia of facial structures and the limb buds. Expression levels of both mRNA and protein decline over the course of development. In the adult brain moderate Phf6 expression is maintained in projection neurons, such as mitral cells in the olfactory bulb, cerebrocortical pyramidal cells and cerebellar Purkinje cells. Phf6 gene expression and nuclear localisation of Phf6 protein correlate with clinical symptoms in BFLS patients, namely mental disability, pan-anterior pituitary hormonal deficiency and facial as well digit abnormalities.

Evaluating cognition in individuals with Huntington disease: Neuro-QoL cognitive functioning measures

Publisher: Springer Science and Business Media LLC

Date: 08-12-2018

DOI: 10.1007/S11136-017-1755-6

University Of Adelaide Faculty Of Health Sciences

Location: Australia

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Women's And Children's Hospital Adelaide

Location: Australia

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Flinders University

Location: Australia

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University Of Adelaide

Location: Australia

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SA Pathology

Location: Australia

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Adelaide Training College Of Complementary Medicine

Location: Australia

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ARC Future Fellowships - Grant ID: FT120100086

Start Date: 12-2012

End Date: 06-2017

Amount: $787,989.00

Funder: Australian Research Council