Aristaless-related Homeobox Mutations Research Articles

Aristaless-Related Homeobox (ARX): Epilepsy Phenotypes beyond Lissencephaly and Brain Malformations

AbstractThe Aristaless-related homeobox (ARX) transcription factor is involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopmental disorders in humans and are responsible for both malformation (in particular lissencephaly) and nonmalformation complex phenotypes. The epilepsy phenotypes related to ARX mutations are West syndrome and X-linked infantile spasms, X-linked myoclonic epilepsy with spasticity and intellectual development and Ohtahara and early infantile epileptic encephalopathy syndrome, which are related in most of the cases to intellectual disability and are often drug resistant. In this article, we shortly reviewed current knowledge of the function of ARX with a particular attention on its consequences in the development of epilepsy during early childhood.

Mutations of ARX and non-syndromic intellectual disability in Chinese population.

Mutations of Aristaless-related homeobox (ARX) gene were looked as the third cause of non-syndromic intellectual disability (NSID), while the boundary between true disease-causing mutations and non-disease-causing variants within this gene remains elusive. To investigate the relationship between ARX mutations and NSID, a panel comprising six reported causal mutations of the ARX was detected in 369 sporadic NSID patients and 550 random participants in Chinese. Two mutations, c.428_451 dup and p.G286S, may be disease-causing mutations for NSID, while p.Q163R and p.P353L showed a great predictive value in female NSID diagnosis with significant associations (X2 = 19.60, p = 9.54e-6 for p.Q163R; X2 = 25.70, p = 4.00e-07 for p.P353L), carriers of these mutations had an increased risk of NSID of more than fourfold. Detection of this panel also predicted significant associations between genetic variants of the ARX gene and NSID (p = 3.73e-4). The present study emphasized the higher genetic burden of the ARX gene on NSID in the Chinese population, molecular analysis of this gene should be considered for patients presenting NSID of unknown etiology.

A new mouse model of ARX dup24 recapitulates the patients' behavioral and fine motor alterations.

The aristaless-related homeobox (ARX) transcription factor is involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopmental disorders in humans, among which the most frequent, a 24 bp duplication in the polyalanine tract 2 (c.428_451dup24), gives rise to intellectual disability, fine motor defects with or without epilepsy. To understand the functional consequences of this mutation, we generated a partially humanized mouse model carrying the c.428_451dup24 duplication (Arxdup24/0) that we characterized at the behavior, neurological and molecular level. Arxdup24/0 males presented with hyperactivity, enhanced stereotypies and altered contextual fear memory. In addition, Arxdup24/0 males had fine motor defects with alteration of reaching and grasping abilities. Transcriptome analysis of Arxdup24/0 forebrains at E15.5 showed a down-regulation of genes specific to interneurons and an up-regulation of genes normally not expressed in this cell type, suggesting abnormal interneuron development. Accordingly, interneuron migration was altered in the cortex and striatum between E15.5 and P0 with consequences in adults, illustrated by the defect in the inhibitory/excitatory balance in Arxdup24/0 basolateral amygdala. Altogether, we showed that the c.428_451dup24 mutation disrupts Arx function with a direct consequence on interneuron development, leading to hyperactivity and defects in precise motor movement control and associative memory. Interestingly, we highlighted striking similarities between the mouse phenotype and a cohort of 33 male patients with ARX c.428_451dup24, suggesting that this new mutant mouse line is a good model for understanding the pathophysiology and evaluation of treatment.

Developmental interneuron subtype deficits after targeted loss of Arx

BackgroundAristaless-related homeobox (ARX) is a paired-like homeodomain transcription factor that functions primarily as a transcriptional repressor and has been implicated in neocortical interneuron specification and migration. Given the role interneurons appear to play in numerous human conditions including those associated with ARX mutations, it is essential to understand the consequences of mutations in this gene on neocortical interneurons. Previous studies have examined the effect of germline loss of Arx, or targeted mutations in Arx, on interneuron development. We now present the effect of conditional loss of Arx on interneuron development.ResultsTo further elucidate the role of Arx in forebrain development we performed a series of anatomical and developmental studies to determine the effect of conditional loss of Arx specifically from developing interneurons in the neocortex and hippocampus. Analysis and cell counts were performed from mouse brains using immunohistochemical and in situ hybridization assays at 4 times points across development. Our data indicate that early in development, instead of a loss of ventral precursors, there is a shift of these precursors to more ventral locations, a deficit that persists in the adult nervous system. The result of this developmental shift is a reduced number of interneurons (all subtypes) at early postnatal and later time periods. In addition, we find that X inactivation is stochastic, and occurs at the level of the neural progenitors.ConclusionThese data provide further support that the role of Arx in interneuron development is to direct appropriate migration of ventral neuronal precursors into the dorsal cortex and that the loss of Arx results in a failure of interneurons to reach the cortex and thus a deficiency in interneurons.

Conditional Loss of Arx From the Developing Dorsal Telencephalon Results in Behavioral Phenotypes Resembling Mild HumanARXMutations

Mutations in the Aristaless-Related Homeobox (ARX) gene cause structural anomalies of the brain, epilepsy, and neurocognitive deficits in children. During forebrain development, Arx is expressed in both pallial and subpallial progenitor cells. We previously demonstrated that elimination of Arx from subpallial-derived cortical interneurons generates an epilepsy phenotype with features overlapping those seen in patients with ARX mutations. In this report, we have selectively removed Arx from pallial progenitor cells that give rise to the cerebral cortical projection neurons. While no discernable seizure activity was recorded, these mice exhibited a peculiar constellation of behaviors. They are less anxious, less social, and more active when compared with their wild-type littermates. The overall cortical thickness was reduced, and the corpus callosum and anterior commissure were hypoplastic, consistent with a perturbation in cortical connectivity. Taken together, these data suggest that some of the structural and behavioral anomalies, common in patients with ARX mutations, are specifically due to alterations in pallial progenitor function. Furthermore, our data demonstrate that some of the neurobehavioral features found in patients with ARX mutations may not be due to on-going seizures, as is often postulated, given that epilepsy was eliminated as a confounding variable in these behavior analyses.

Mutational screening of ARX gene in Iranian families with X-linked intellectual disability.

Mutations in the human aristaless-related homeobox (ARX) gene are amongst the major causes of developmental and neurological disorders. They are responsible for a wide spectrum of phenotypes, including nonsyndromic X-linked intellectual disability (NS-XLID), and syndromic (XLIDS) forms such as X-linked lissencephaly with abnormal genitalia (XLAG), Partington syndrome (PRTS), and X-linked infantile spasm syndrome (ISSX). The recurrent 24 bp duplication mutation, c.428_451dup(24 bp), is the most frequent ARX mutation, which accounts for ~40% of all cases reported to date. We have screened the entire coding sequences of the ARX gene in 65 Iranian families with intellectual disabilities in order to obtain the relative prevalence of ARX mutations. At first these families were screened for the most recurrent mutation, the c.428_451dup(24 bp). For samples with negative results, single strand conformation polymorphism (SSCP) analysis was performed. We identified one family with the c.428_451dup(24 bp) duplication. Three shifts (one shift in exon 5 and two shifts in exon 4) were also identified among the total families. According to the results of the sequencing analysis, two shifts were not associated with any mutation and the other one was a c.1347C>T (p.G449G) substitution in exon 4. Hence, we suggest that molecular analysis of ARX mutations as a second cause of XLID should be considered as routine diagnostic procedure in any male who presents with either NS-XLID or XLIDS.

Is there a Mendelian transmission ratio distortion of the c.429_452dup(24bp) polyalanine tract ARX mutation?

Intellectual disability is common. Aristaless-related homeobox (ARX) gene is one of the most frequently mutated and pleiotropic genes, implicated in 10 different phenotypes. More than half of ~100 reported cases with ARX mutations are due to a recurrent duplication of 24 bp, c.429_452dup, which leads to polyalanine tract expansion. The excess of affected males among the offspring of the obligate carrier females raised the possibility of transmission ratio distortion for the c.429_452dup mutation. We found a significant deviation from the expected Mendelian 1:1 ratio of transmission in favour of the c.429_452dup ARX mutation. We hypothesise that the preferential transmission of the c.429_452dup mutation may be due to asymmetry of meiosis in the oocyte. Our findings may have implications for genetic counselling of families segregating the c.429_452dup mutation and allude to putative role of ARX in oocyte biology.

High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies

Genetic investigations of X-linked intellectual disabilities have implicated the ARX (Aristaless-related homeobox) gene in a wide spectrum of disorders extending from phenotypes characterised by severe neuronal migration defects such as lissencephaly, to mild or moderate forms of mental retardation without apparent brain abnormalities but with associated features of dystonia and epilepsy. Analysis of Arx spatio-temporal localisation profile in mouse revealed expression in telencephalic structures, mainly restricted to populations of GABAergic neurons at all stages of development. Furthermore, studies of the effects of ARX loss of function in humans and animal models revealed varying defects, suggesting multiple roles of this gene during brain development. However, to date, little is known about how ARX functions as a transcription factor and the nature of its targets. To better understand its role, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified a total of 1006 gene promoters bound by Arx in transfected neuroblastoma (N2a) cells and in mouse embryonic brain. Approximately 24% of Arx-bound genes were found to show expression changes following Arx overexpression or knock-down. Several of the Arx target genes we identified are known to be important for a variety of functions in brain development and some of them suggest new functions for Arx. Overall, these results identified multiple new candidate targets for Arx and should help to better understand the pathophysiological mechanisms of intellectual disability and epilepsy associated with ARX mutations.

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

ARX mutations cause a diverse 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 samples from 613 individuals 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 samples 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.

Aristaless‐related homeobox mutations

SummaryMutations in the Aristaless‐related homeobox gene (ARX) are linked to infantile spasms and other developmental epilepsies. How loss of Arx leads to epilepsy is not well understood. This review will discuss the spectrum of Arx disorders and the emerging evidence, from animal models, of the function of Arx during development and the potential role in generating an epilepsy phenotype. For an expanded treatment of this topic see Jasper’s Basic Mechanisms of the Epilepsies, Fourth Edition (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado‐Escueta AV, eds) published by Oxford University Press (available on the National Library of Medicine Bookshelf [NCBI] at http://www.ncbi.nlm.nih.gov/books).

Westward Ho! Pioneering Mouse Models for X-Linked Infantile Spasms Syndrome

Targeted Loss of Arx Results in a Developmental Epilepsy Mouse Model and Recapitulates the Human Phenotype in Heterozygous Females. Marsh E, Fulp C, Gomez E, Nasrallah I, Minarcik J, Sudi J, Christian SL, Mancini G, Labosky P, Dobyns W, Brooks-Kayal A, Golden JA. Brain 2009;132(Pt 6):1563–1576. Mutations in the X-linked aristaless-related homeobox gene (ARX) have been linked to structural brain anomalies as well as multiple neurocognitive deficits. The generation of Arx-deficient mice revealed several morphological anomalies, resembling those observed in patients and an interneuron migration defect but perinatal lethality precluded analyses of later phenotypes. Interestingly, many of the neurological phenotypes observed in patients with various ARX mutations can be attributed, in part, to interneuron dysfunction. To directly test this possibility, mice carrying a floxed –Arx allele were generated and crossed to Dlx5/6 CRE-IRES-GFP( Dlx5/6 CIG ) mice, conditionally deleting Arx from ganglionic eminence derived neurons including cortical interneurons. We now report that Arx- /y; Dlx5/6 CIG (male) mice exhibit a variety of seizure types beginning in early-life, including seizures that behaviourally and electroencephalographically resembles infantile spasms, and show evolution through development. Thus, this represents a new genetic model of a malignant form of paediatric epilepsy, with some characteristics resembling infantile spasms, caused by mutations in a known infantile spasms gene. Unexpectedly, approximately half of the female mice carrying a single mutant Arx allele ( Arx-/+; Dlx5/6 CIG) also developed seizures. We also found that a subset of human female carriers have seizures and neurocognitive deficits. In summary, we have identified a previously unrecognized patient population with neurological deficits attributed to ARX mutations that are recapitulated in our mouse model. Furthermore, we show that perturbation of interneuron subpopulations is an important mechanism underling the pathogenesis of developmental epilepsy in both hemizygous males and carrier females. Given the frequency of ARX mutations in patients with infantile spasms and related disorders, our data unveil a new model for further understanding the pathogenesis of these disorders. A Triplet Repeat Expansion Genetic Mouse Model of Infantile Spasms Syndrome, Arx(GCG)10+7, with Interneuronopathy, Spasms in Infancy, Persistent Seizures, and Adult Cognitive and Behavioral Impairment. Price MG, Yoo JW, Burgess DL, Deng F, Hrachovy RA, Frost JD Jr, Noebels JL. J Neurosci 2009;29(27):8752–8763. Infantile spasms syndrome (ISS) is a catastrophic pediatric epilepsy with motor spasms, persistent seizures, mental retardation, and in some cases, autism. One of its monogenic causes is an insertion mutation [c.304ins (GCG)7] on the X chromosome, expanding the first polyalanine tract of the interneuron-specific transcription factor Aristaless-related homeobox (ARX) from 16 to 23 alanine codons. Null mutation of the Arx gene impairs GABA and cholinergic interneuronal migration but results in a neonatal lethal phenotype. We developed the first viable genetic mouse model of ISS that spontaneously recapitulates salient phenotypic features of the human triplet repeat expansion mutation. Arx(GCG)10+7 (“ Arx plus 7”) pups display abnormal spasm-like myoclonus and other key EEG features, including multifocal spikes, electrodecremental episodes, and spontaneous seizures persisting into maturity. The neurobehavioral profile of Arx mutants was remarkable for lowered anxiety, impaired associative learning, and abnormal social interaction. Laminar decreases of Arx+ cortical interneurons and a selective reduction of calbindin-, but not parvalbumin- or calretinin-expressing interneurons in neo-cortical layers and hippocampus indicate that specific classes of synaptic inhibition are missing from the adult forebrain, providing a basis for the seizures and cognitive disorder. A significant reduction of calbindin-, NPY (neuropeptide Y)-expressing, and cholinergic interneurons in the mutant striatum suggest that dysinhibition within this network may contribute to the dyskinetic motor spasms. This mouse model narrows the range of critical pathogenic elements within brain inhibitory networks essential to recreate this complex neurodevelopmental syndrome.

Identification of Arx transcriptional targets in the developing basal forebrain

Mutations in the aristaless-related homeobox (ARX) gene are associated with multiple neurologic disorders in humans. Studies in mice indicate Arx plays a role in neuronal progenitor proliferation and development of the cerebral cortex, thalamus, hippocampus, striatum, and olfactory bulbs. Specific defects associated with Arx loss of function include abnormal interneuron migration and subtype differentiation. How disruptions in ARX result in human disease and how loss of Arx in mice results in these phenotypes remains poorly understood. To gain insight into the biological functions of Arx, we performed a genome-wide expression screen to identify transcriptional changes within the subpallium in the absence of Arx. We have identified 84 genes whose expression was dysregulated in the absence of Arx. This population was enriched in genes involved in cell migration, axonal guidance, neurogenesis, and regulation of transcription and includes genes implicated in autism, epilepsy, and mental retardation; all features recognized in patients with ARX mutations. Additionally, we found Arx directly repressed three of the identified transcription factors: Lmo1, Ebf3 and Shox2. To further understand how the identified genes are involved in neural development, we used gene set enrichment algorithms to compare the Arx gene regulatory network (GRN) to the Dlx1/2 GRN and interneuron transcriptome. These analyses identified a subset of genes in the Arx GRN that are shared with that of the Dlx1/2 GRN and that are enriched in the interneuron transcriptome. These data indicate Arx plays multiple roles in forebrain development, both dependent and independent of Dlx1/2, and thus provides further insights into the understanding of the mechanisms underlying the pathology of mental retardation and epilepsy phenotypes resulting from ARX mutations.

Mutation screening of the Aristaless-related homeobox ( ARX) gene in Thai pediatric patients with delayed development: First report from Thailand

Mutations in the Aristaless-related homeobox gene, ARX, have been a cause of X-linked mental retardation (XLMR) and are responsible for a vast phenotypic spectrum including syndromic and non-syndromic forms of mental retardation. Since the gene was initially identified, it has been generally screened in several patients with XLMR. This study is the first report of ARX mutational screening in Thai pediatric patients with delayed development. Two hundred and fifty-one patients participated in this study. Two hundred and three of the 251 patients were initially referred for molecular diagnosis of the Fragile XA syndrome and had negative test results. The remaining 48 patients were specifically recruited for the ARX mutational analysis and had previously reported phenotypes of the ARX mutations. Screening for the c.428_451 dup mutation was performed in all samples. Screening for other point mutations in all coding exons was performed in all 48 patients recruited for the ARX mutational analysis and in 29 patients initially referred for diagnosis of the Fragile XA syndrome who had two or more affected males in the family suggesting an X-linked inheritance pattern. Two patients were found to have the c.428_451 dup mutation. Considering genotype-phenotype correlation, we suggest screening of the most common mutation, the c.428_451 dup mutation by PCR, in patients with infantile spasm syndrome, Partington syndrome and non-syndromic X-linked mental retardation. Screening in patients who have negative Fragile XA test results should be considered when no other known causes of mental retardation are identified especially in families with suggestive X-linked inheritance pattern.

Mutation screening of the ARX gene in patients with autism

Mutations in the Aristaless related homeobox (ARX) gene are associated with a broad spectrum of disorders, including nonsyndromic X-linked mental retardation, sometimes associated with epilepsy, as well as syndromic forms with brain abnormalities and abnormal genitalia. Furthermore, ARX mutations have been described in a few patients with autism or autistic features. In this study, we screened the ARX gene in 226 male patients with autism spectrum disorders and mental retardation; 42 of the patients had epilepsy. The mutation analysis was performed by direct sequencing of all exons and flanking regions. No ARX mutations were identified in any of the patients tested. These findings indicate that mutations in the ARX gene are very rare in autism.

Neuroanatomical distribution of ARX in brain and its localisation in GABAergic neurons

Recent human genetics approaches identified the Aristaless-related homeobox ( ARX) gene as the causative gene in X-linked infantile spasms, Partington syndrome, and non-syndromic mental retardation as well as in forms of lissencephaly with abnormal genitalia. The ARX predicted protein belongs to a large family of homeoproteins and is characterised by a C-terminal Aristaless domain and an octapeptide domain near the N-terminus. In order to learn more about ARX function, we have studied in detail Arx expression in the central nervous system during mouse embryonic development as well as in the adult. During early stages of development, Arx is expressed in a significant proportion of neurons in the cortex, the striatum, the ganglionic eminences and also in the spinal cord. In the adult, expression of Arx is still present and restricted to regions that are known to be rich in GABAergic neurons such as the amygdala and the olfactory bulb. A possible role for Arx in this type of neurons is further reinforced by the expression of Arx in a subset of GABAergic interneurons in young and mature primary cultures of cortical neuronal cells as well as in vivo. Moreover, these data could explain the occurrence of seizures in the great majority of patients with an ARX mutation, due to mislocalisation or dysfunction of GABAergic neurons. We also performed ARX wild-type and mutant over-expression experiments and found that the different ARX mutations tested did not modify the morphology of the cells. Moreover, no abnormal cell death or protein aggregation was observed, hence suggesting that more subtle pathogenic mechanisms are involved.