Aristaless-related Homeobox Gene Research Articles

ARX regulates cortical interneuron differentiation and migration.

Mutations in aristaless-related homeobox ( ARX ) are associated with neurodevelopmental disorders including developmental epilepsies, intellectual disabilities, and autism spectrum disorders, with or without brain malformations. Aspects of these disorders have been linked to abnormal cortical interneuron (cIN) development and function. To further understand ARX's role in cIN development, multiple Arx mutant mouse lines were interrogated. We found that ARX is critical for controlling cIN numbers and distribution, especially, in the developing marginal zone (MZ). Single cell transcriptomics and ChIP-seq, combined with functional studies, revealed ARX directly or indirectly regulates genes involved in proliferation and the cell cycle (e.g., Bub3 , Cspr3 ), fate specification (e.g., Nkx2.1 , Maf , Mef2c ), and migration (e.g., Nkx2.1 , Lmo1 , Cxcr4 , Nrg1 , ErbB4 ). Our data suggest that the MZ stream defects primarily result from disordered cell-cell communication. Together our findings provide new insights into the mechanisms underlying cIN development and migration and how they are disrupted in several disorders.

Type 1 early infantile epileptic encephalopathy: A case report and literature review.

Variants in the Aristaless-related homeobox (ARX) gene lead to a variety of phenotypes, with intellectual disability being a steady feature. Other features can include severe epilepsy, spasticity, movement disorders, hydranencephaly, and ambiguous genitalia in males. X-linked Ohtahara syndrome or Type 1 early infantile epileptic encephalopathy (EIEE1) is a severe early-onset epileptic encephalopathy with arrested psychomotor development caused by hemizygous mutations in the ARX gene, which encodes a transcription factor in fundamental brain developmental processes. We presented a case report of a 2-year-old boy who exhibited symptoms such as microcephaly, seizures, and severe multifocal epileptic abnormalities, and genetic techniques such as autozygosity mapping, Sanger sequencing, and whole-exome sequencing. We confirmed that the patient had the NM_139058.3:c.84C>A; p.(Cys28Ter) mutation in the ARX gene. The patient with EIEE1 had physical symptoms and hypsarrhythmia on electroencephalogram. Genetic testing identified a causative mutation in the ARX gene, emphasizing the role of genetic testing in EIEE diagnosis.

EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells.

Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.

Comment on: Are Parenchyma-Sparing Resections Really Appropriate for Small (

Comment on: Are Parenchyma-Sparing Resections Really Appropriate for Small (<3 cm) Nonfunctional Pancreatic Neuroendocrine Tumors?

Deregulation of microtubule organization and RNA metabolism in Arx models for lissencephaly and developmental epileptic encephalopathy.

X-linked lissencephaly with abnormal genitalia (XLAG) and developmental epileptic encephalopathy-1 (DEE1) are caused by mutations in the Aristaless-related homeobox (ARX) gene, which encodes a transcription factor responsible for brain development. It has been unknown whether the phenotypically diverse XLAG and DEE1 phenotypes may converge on shared pathways. To address this question, a label-free quantitative proteomic approach was applied to the neonatal brain of Arx knockout (ArxKO/Y) and knock-in polyalanine (Arx(GCG)7/Y) mice that are respectively models for XLAG and DEE1. Gene ontology and protein–protein interaction analysis revealed that cytoskeleton, protein synthesis and splicing control are deregulated in an allelic-dependent manner. Decreased α-tubulin content was observed both in Arx mice and Arx/alr-1(KO) Caenorhabditis elegans ,and a disorganized neurite network in murine primary neurons was consistent with an allelic-dependent secondary tubulinopathy. As distinct features of Arx(GCG)7/Y mice, we detected eIF4A2 overexpression and translational suppression in cortex and primary neurons. Allelic-dependent differences were also established in alternative splicing (AS) regulated by PUF60 and SAM68. Abnormal AS repertoires in Neurexin-1, a gene encoding multiple pre-synaptic organizers implicated in synaptic remodelling, were detected in Arx/alr-1(KO) animals and in Arx(GCG)7/Y epileptogenic brain areas and depolarized cortical neurons. Consistent with a conserved role of ARX in modulating AS, we propose that the allelic-dependent secondary synaptopathy results from an aberrant Neurexin-1 repertoire. Overall, our data reveal alterations mirroring the overlapping and variant effects caused by null and polyalanine expanded mutations in ARX. The identification of these effects can aid in the design of pathway-guided therapy for ARX endophenotypes and NDDs with overlapping comorbidities.

Generation of an induced pluripotent stem cell line from an Ohtahara syndrome patient with the hemizygous mutation p.Q503Afs*28 (c.1507_1508del) in the ARX gene

Aristaless-related homeobox (ARX)-related disorders are recessive X-linked intellectual disability disorders. We encountered a patient with a hemizygous mutation (c.1507_1508del) showing intellectual disability, early-onset epileptic encephalopathy and Ohtahara syndrome. The patient had female genitals, but an XY karyotype. We established an induced pluripotent stem cell (iPSC) line from the peripheral blood mononuclear cells (PBMCs) of a six-month Chinese child with a hemizygous mutation (c.1507_1508del) in ARX. The PBMCs were reprogrammed with Sendai viral vectors. The iPSCs showed stable amplification, pluripotency-related gene expression, and trilineage differentiation potential. Karyotype analysis of the iPSCs showed 23 pairs of chromosomes with normal structure and sex chromosome is XY.

Non-functional pancreatic neuroendocrine tumours: ATRX/DAXX and alternative lengthening of telomeres (ALT) are prognostically independent from ARX/PDX1 expression and tumour size

ObjectiveRecent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic...

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

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.

The expression of aristaless-related homeobox in neural progenitors of gyrencephalic carnivore ferrets

Aristaless-related homeobox (ARX) has important functions in the development of various organs including the brain. Mutations of the human ARX gene have been associated with malformations of the cerebral cortex such as microcephaly and lissencephaly. Although the expression patterns of ARX in the lissencephalic cerebral cortex of mice have been intensively investigated, those in expanded gyrencephalic brains remained unclear. Here, we show the expression patterns of ARX in the developing cerebral cortex of gyrencephalic carnivore ferrets. We found that ARX was expressed not only in intermediate progenitor (IP) cells but also in outer radial glial (oRG) cells, which are neural progenitors preferentially observed in the gyrencephalic cerebral cortex. We found that the majority of ARX-positive oRG cells expressed the proliferating cell marker Ki-67. These results may indicate that ARX in oRG cells mediates the expansion of the gyrencephalic cerebral cortex during development and evolution.

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

The need to interpret thepathogenicity of novel missense variants of unknown significance identified in the homeodomain of X-chromosome aristaless-related homeobox (ARX) gene prompted us to assess the utility of conservation and constraint across these domains in multiple genes compared to conventional in vitro functional analysis. Pathogenic missense variants clustered in the homeodomain of ARX contribute to intellectual disability (ID) and epilepsy, with and without brain malformation in affected males. Here we report novel c.1112G>A, p.Arg371Gln and c.1150C>T, p.Arg384Cys variants in male patients with ID and severe seizures. The third case of a male patient with a c.1109C>T, p.Ala370Val variant is perhaps the first example of ID and autism spectrum disorder (ASD), without seizures or brain malformation. We compiled data sets of pathogenic variants from ClinVar and presumed benign variation from gnomAD and demonstrated that the high levels of sequence conservation and constraint of benign variation within the homeodomain impacts upon the ability of publicly available in silico prediction tools to accurately discern likely benign from likely pathogenic variants in these data sets. Despite this, considering the inheritance patterns of the genes and disease variants with the conservation and constraint of disease variants affecting the homeodomain in conjunction with current clinical assessments may assist in predicting the pathogenicity of missense variants, particularly for genes with autosomal recessive and X-linked patterns of disease inheritance, such as ARX. In vitro functional analysis demonstrates that the transcriptional activity of all three variants wasdiminished compared to ARX-Wt. We review the associated phenotypes of the published cases of patients with ARX homeodomain variants and propose expansion of the ARX-related phenotype to include severe ID and ASD without brain malformations or seizures. We propose that the use of the constraint and conservation data in conjunction with consideration of the patient phenotype and inheritance pattern may negate the need for the experimental functional validation currently required to achieve a diagnosis.

Novel ARX mutation identified in infantile spasm syndrome patient

We report a 7-year-old boy with infantile spasms caused by a novel mutation in the Aristaless-related homeobox (ARX) gene. He showed infantile spasms and hypsarrhythmia on electroencephalogram from early infancy. Brain MRI did not reveal severe malformation of the brain except mild hypoplasia of the corpus callosum. Two-fold adrenocorticotropic hormone (ACTH) therapy failed to control the seizures, and ketogenic diet therapy and multi-antiepileptic drug therapy were required as he showed intractable daily tonic-clonic seizures. Exome sequencing identified a hemizygous mutation in the ARX gene, NG_008281.1(ARX_v001):c.1448 + 1 G > A, chrX: 25025227 C > T (GRCh37). To our knowledge, this mutation has not been reported previously.

Dynamic causal modeling of hippocampal activity measured via mesoscopic voltage-sensitive dye imaging

The aim of this paper is to present a dynamic causal modeling (DCM) framework for hippocampal activity measured via voltage-sensitive dye imaging (VSDI). We propose a DCM model of the hippocampus that summarizes interactions between the hilus, CA3 and CA1 regions. The activity of each region is governed via a neuronal mass model with two inhibitory and one/two excitatory neuronal populations, which can be linked to measurement VSDI by scaling neuronal activity. To optimize the model structure for the hippocampus, we propose two Bayesian schemes: Bayesian hyperparameter optimization to estimate the unknown electrophysiological properties necessary for constructing a mesoscopic hippocampus model; and Bayesian model reduction to determine the parameterization of neural properties, and to test and include potential connections (morphologically inferred without direct evidence yet) in the model by evaluating group-level model evidence. The proposed method was applied to model spatiotemporal patterns of accumulative responses to consecutive stimuli in separate groups of wild-type mice and epileptic aristaless-related homeobox gene (Arx) conditional knock-out mutant mice (Arx−/+;Dlx5/6CRE−IRES-GFP) in order to identify group differences in the effective connectivity within the hippocampus. The causal role of each group-differing connectivity in generating mutant-like responses was further tested. The group-level analysis identified altered intra- and inter-regional effective connectivity, some of which are crucial for explaining mutant-like responses. The modelling results for the hippocampal activity suggest the plausibility of the proposed mesoscopic hippocampus model and the usefulness of utilizing the Bayesian framework for model construction in the mesoscale modeling of neural interactions using DCM.

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

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 individuals. 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.

ARX gene with an impressive role in X-linked intellectual disability

Intellectual disability is the most common neurodevelopmental defect in the worldwide. X-linked intellectual disability (XLID) is the frequent form of intellectual disability which includes a heterogeneous group of inherited disorders emerging as various degrees of intellectual disabilities. XLID has a prevalence of 2.6 cases per 1,000 in the general population and accounts for over 10% of all cases of intellectual disability. Based on associated phenotypes, XLID is subdivided into syndromic (S-XLID) and non-syndromic (NS-XLID) forms; where two third of XLID cases are thought to be non-syndromic. Among the non-syndromic form, the aristalessrelated homeobox gene (ARX) gene is one of the ideal candidates to be evaluated in NS-XLID, since its mutations are responsible for about 9.5% of XLID cases. The ARX is located on the Xp22.13 genomic region and encodes a highly conserved protein with a considerable role in Wnt/β-catenin signaling pathway. Base on review literature, mutations in ARX gene has a particular influence on the critical processes associated with the brain development. Our results in bioinformatics study of molecular features, second and quaternary structures of ARX gene and also the phylogeny tree of ARX protein is showed that the ARX is a highly conserved protein with a substantial role in an important developmental pathway and its deficiency can cause irreversible defects, mainly in brain, that leads to the development of XLID as a common form of intellectual disability and also, the sequence alignment of this protein with other spices confirms that the functional domains of ARX protein are highly conserved, thus it has been predicted that the mutations of this gene is highly pathogenic. Alongside, we mainly focused to gather the data addressing the structural properties of ARX protein and bioinformatics assay of this protein to find the important role of ARX gene in the integrity of normal brain development.

Present status and expectation of aristaless-related homeobox (ARX) in endocrine pancreas.

The aristaless-related homeobox (ARX) gene has become one of most frequently mutated genes which is closely linked with development of the vertebrate central nervous system; however, the molecular and clinical bases of its function in the proliferation and differentiation of the endocrine pancreas have not, to date, been systematically characterized. ARX is considered as a regulator which determines endocrine cell fate and a bio-marker of the pancreatic α-cell. Disruption and mutation of ARX are found to lead to the deletion and reduction of α-cells both in mice models and in humans. Furthermore, expression of ARX is regulated by multiple transcription factors involved in development of the pancreas, such as Ngn3, Isl1, Nkx2.2 and Nkx6.1. Taken together, given the vital importance of glucagon in diabetes treatment, it is possible that ARX may down-regulate exorbitant glucagon levels by reducing the number of α-cells as a direct target; thus, the role of ARX in the maintenance of α-cell identity and quantity should be investigated and summarized. This article mainly focuses on the role of ARX in the endocrine pancreas, introduces the ARX-related animal model and transcription factors, and highlights the latest advances in our understanding in order to provide a clearer theoretical foundation for future scientific research.

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

Aristaless-related homeobox (ARX) gene encodes a paired-type homeodomain transcription factor with critical roles in development. Here we identify that ARX protein is phosphorylated. Using mass spectrometry and in vitro kinase assays we identify phosphorylation at serines 37, 67 and 174. Through yeast-2-hybrid and CoIP we identified PICK1 (Protein interacting with C kinase 1) binding with the C-terminal region of ARX. PICK1 is a scaffold protein known to facilitate phosphorylation of protein partners by protein kinase C alpha (PRKCA). We confirm that ARX is phosphorylated by PRKCA and demonstrate phosphorylation at serine 174. We demonstrate that phosphorylation is required for correct transcriptional activity of the ARX protein using transcriptome-wide analysis of gene expression of phospho-null mutants (alanines replacing serines) compared to ARX wild-type (ARX-WT) overexpressed in pancreatic alpha TC cells. Compared to untransfected cells, ARX-WT overexpression significantly altered expression of 70 genes (Log2FC >+/-1.0, P-value <0.05). There were fewer genes with significantly altered expression compared to untransfected cells with the double phospho-null mutant Ser37Ala+Ser67Ala (26%) and Ser174Ala (39%), respectively. We demonstrate that the c-terminal region of ARX required to bind PICK1 causes a shift in PICK1 subcellular localisation to the nucleus to co-locate with the ARX protein, and truncation of this C-terminal region leads to the same loss of transcriptional activation as S174A mutant. In conclusion, we show that ARX is phosphorylated at several sites and that this modification affects its transcriptional activity.

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.

Status dystonicus – Undefined or undefinable?

In this issue of the journal, Gorman and Colleagues add to the steadily growing catalogue of reports of status dystonicus (SD) in childhood. 1 Gorman K.M. Cary H. Gaffney L. et al. Status dystonicus due to missense variant in ARX: diagnosis and management. Eur J Paediatr Neurol EJPN Offic J Eur Paediatr Neurol Soc. 2018; ([Epub ahead of print]) Abstract Full Text Full Text PDF Scopus (5) Google Scholar Their case is notable, describing as it does a child with a missense variant in the aristaless domain of the Aristaless-related homeobox gene (ARX), a domain of the gene not previously associated with SD. The case also describes all of the classical features SD that the clinical community have come to recognise, including the initial diagnostic confusion with status epilepticus (SE), and a complex and prolonged course of pharmacological management.

Status dystonicus due to missense variant in ARX: Diagnosis and management

Movement disorders are increasingly identified in infantile encephalopathies due to single gene disorders (e.g. SCN2A, CDKL5, ARX). The associated movement disorder can be challenging to recognise and treat. We report a 2 year-old boy with a background history of Ohtahara syndrome due to a missense variant in ARX (the aristaless-related homeobox gene) who subsequently developed status dystonicus. ARX is a transcription factor that plays a critical role in cortical neuronal development and is associated with a range of important neurodevelopmental disorders depending on the site of the pathogenic variant. Cases of status dystonicus are described with variants affecting the polyalanine expansion region of ARX but have not been reported previously with variants affecting the aristaless domain of ARX as in this case. Dystonic episodes posed a challenge in recognition and treatment, including confusion with status epilepticus. We discuss the difficulties in diagnosis and management of status dystonicus, an underreported life-threatening emergency in children.

Basal ganglia involvement in ARX patients: The reason for ARX patients very specific grasping?

The ARX (Aristaless Related homeoboX) gene was identified in 2002 as responsible for XLAG syndrome, a lissencephaly characterized by an almost complete absence of cortical GABAergic interneurons, and for milder forms of X-linked Intellectual Disability (ID) without apparent brain abnormalities. The most frequent mutation found in the ARX gene, a duplication of 24 base pairs (c.429_452dup24) in exon 2, results in a recognizable syndrome in which patients present ID without primary motor impairment, but with a very specific upper limb distal motor apraxia associated with a pathognomonic hand-grip, described as developmental Limb Kinetic Apraxia (LKA).In this study, we first present ARX expression during human fetal brain development showing that it is strongly expressed in GABAergic neuronal progenitors during the second and third trimester of pregnancy. We show that although ARX expression strongly decreases towards the end of gestation, it is still present after birth in some neurons of the basal ganglia, thalamus and cerebral cortex, suggesting that ARX also plays a role in more mature neuron functioning. Then, using morphometric brain MRI in 13 ARX patients carrying c.429_452dup24 mutation and in 13 sex- and age-matched healthy controls, we show that ARX patients have a significantly decreased volume of several brain structures including the striatum (and more specifically the caudate nucleus), hippocampus and thalamus as well as decreased precentral gyrus cortical thickness. We observe a significant correlation between caudate nucleus volume reduction and motor impairment severity quantified by kinematic parameter of precision grip.As basal ganglia are known to regulate sensorimotor processing and are involved in the control of precision gripping, the combined decrease in cortical thickness of primary motor cortex and basal ganglia volume in ARX dup24 patients is very likely the anatomical substrate of this developmental form of LKA.