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1.  Identification of Arx transcriptional targets in the developing basal forebrain 
Human Molecular Genetics  2008;17(23):3740-3760.
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.
doi:10.1093/hmg/ddn271
PMCID: PMC2581427  PMID: 18799476
2.  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 
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 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 Plus7”) 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 neocortical 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-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.
doi:10.1523/JNEUROSCI.0915-09.2009
PMCID: PMC2782569  PMID: 19587282
migration; interneuron; GCG repeat mutation; epilepsy; autism; transcription factor
3.  Interneuron, Interrupted: Molecular Pathogenesis of ARX Mutations and X-linked Infantile Spasms 
Current opinion in neurobiology  2012;22(5):859-865.
X-linked Infantile Spasms Syndrome (ISSX) is a catastrophic epilepsy of early childhood with intractable seizures, intellectual disability, and poor prognosis. A spectrum of mutations in the Aristaless-Related Homeobox gene (ARX) has been linked to ISSX, and downstream targets of this interneuron-expressed transcription factor are being defined. Recent advances combining in vitro and in vivo methods have unveiled complex interactions between Arx and its binding partners and their effects on cell migration and maturation that can help explain the diversity of ARX phenotypes. New mutant mouse models of Arx-induced pathology, including a recent human triplet-repeat expansion mutation with a phenotype of infantile spasms and electrographic seizures, provide valuable tools for exploring the pathophysiology of Arx and substrates for testing novel therapies.
doi:10.1016/j.conb.2012.04.006
PMCID: PMC3437236  PMID: 22565167
4.  Arx Polyalanine Expansion in Mice Leads to Reduced Pancreatic α-Cell Specification and Increased α-Cell Death 
PLoS ONE  2013;8(11):e78741.
ARX/Arx is a homeodomain-containing transcription factor necessary for the specification and early maintenance of pancreatic endocrine α-cells. Many transcription factors important to pancreas development, including ARX/Arx, are also crucial for proper brain development. Although null mutations of ARX in human patients result in the severe neurologic syndrome XLAG (X-linked lissencephaly associated with abnormal genitalia), the most common mutation is the expansion of the first polyalanine tract of ARX, which results primarily in the clinical syndrome ISSX (infantile spasms). Mouse models of XLAG, ISSX and other human ARX mutations demonstrate a direct genotype-phenotype correlation in ARX-related neurologic disorders. Furthermore, mouse models utilizing a polyalanine tract expansion mutation have illustrated critical developmental differences between null mutations and expansion mutations in the brain, revealing context-specific defects. Although Arx is known to be required for the specification and early maintenance of pancreatic glucagon-producing α-cells, the consequences of the Arx polyalanine expansion on pancreas development remain unknown. Here we report that mice with an expansion mutation in the first polyalanine tract of Arx exhibit impaired α-cell specification and maintenance, with gradual α-cell loss due to apoptosis. This is in contrast to the re-specification of α-cells into β- and δ-cells that occurs in mice null for Arx. Overall, our analysis of an Arx polyalanine expansion mutation on pancreatic development suggests that impaired α-cell function might also occur in ISSX patients.
doi:10.1371/journal.pone.0078741
PMCID: PMC3827280  PMID: 24236044
5.  Aristaless-Related Homeobox Plays a Key Role in Hyperplasia of the Pancreas Islet α–Like Cells in Mice Deficient in Proglucagon-Derived Peptides 
PLoS ONE  2013;8(5):e64415.
Defects in glucagon action can cause hyperplasia of islet α-cells, however, the underlying mechanisms remain largely to be elucidated. Mice homozygous for a glucagon-GFP knock-in allele (Gcggfp/gfp) completely lack proglucagon-derived peptides and exhibit hyperplasia of GFP-positive α-like cells. Expression of the transcription factor, aristaless-related homeobox (ARX), is also increased in the Gcggfp/gfp pancreas. Here, we sought to elucidate the role of ARX in the hyperplasia of α-like cells through analyses of two Arx mutant alleles (ArxP355L/Y and Arx [330insGCG]7/Y) that have different levels of impairment of their function. Expression of Gfp and Arx genes was higher and the size and number of islets increased in the Gcggfp/gfp pancreas compared to and Gcggfp/+ pancreas at 2 weeks of age. In male Gcggfp/gfp mice that are hemizygous for the ArxP355L/Y mutation that results in a protein with a P355L amino acid substitution, expression of Gfp mRNA in the pancreas was comparable to that in control Gcggfp/+Arx+/Y mice. The increases in islet size and number were also reduced in these mice. Immunohistochemical analysis showed that the number of GFP-positive cells was comparable in Gcggfp/gfp ArxP355L/Y and Gcggfp/+Arx+/Y mice. These results indicate that the hyperplasia is reduced by introduction of an Arx mutation. ArxP355L/Y mice appeared to be phenotypically normal; however, Arx [330insGCG]7/Y mice that have a mutant ARX protein with expansion of the polyalanine tract had a reduced body size and shortened life span. The number of GFP positive cells was further reduced in the Gcggfp/gfp Arx [330insGCG]7/Y mice. Taken together, our findings show that the function of ARX is one of the key modifiers for hyperplasia of islet α-like cells in the absence of proglucagon-derived peptides.
doi:10.1371/journal.pone.0064415
PMCID: PMC3650067  PMID: 23671715
6.  Arx Is a Direct Target of Dlx2 and Thereby Contributes to the Tangential Migration of GABAergic Interneurons 
The Journal of Neuroscience  2008;28(42):10674-10686.
The Arx transcription factor is expressed in the developing ventral telencephalon and subsets of its derivatives. Mutation of human ARX ortholog causes neurological disorders including epilepsy, lissencephaly, and mental retardation. We have isolated the mouse Arx endogenous enhancer modules that control its tightly compartmentalized forebrain expression. Interestingly, they are scattered downstream of its coding region and partially included within the introns of the downstream PolA1 gene. These enhancers are ultraconserved noncoding sequences that are highly conserved throughout the vertebrate phylum. Functional characterization of the Arx GABAergic enhancer element revealed its strict dependence on the activity of Dlx transcription factors. Dlx overexpression induces ectopic expression of endogenous Arx and its isolated enhancer, whereas loss of Dlx expression results in reduced Arx expression, suggesting that Arx is a key mediator of Dlx function. To further elucidate the mechanisms involved, a combination of gain-of-function studies in mutant Arx or Dlx tissues was pursued. This analysis provided evidence that, although Arx is necessary for the Dlx-dependent promotion of interneuron migration, it is not required for the GABAergic cell fate commitment mediated by Dlx factors. Although Arx has additional functions independent of the Dlx pathway, we have established a direct genetic relationship that controls critical steps in the development of telencephalic GABAergic neurons. These findings contribute elucidating the genetic hierarchy that likely underlies the etiology of a variety of human neurodevelopmental disorders.
doi:10.1523/JNEUROSCI.1283-08.2008
PMCID: PMC3844830  PMID: 18923043
basal forebrain; development; epilepsy; GABAergic neuron; neuronal progenitor cell; basal ganglia
7.  Dysgenesis of Enteroendocrine Cells in Aristaless-Related Homeobox Polyalanine Expansion Mutations 
ABSTRACT
Objectives:
Severe congenital diarrhea occurs in approximately half of patients with Aristaless-Related Homeobox (ARX) null mutations. The cause of this diarrhea is unknown. In a mouse model of intestinal Arx deficiency, the prevalence of a subset of enteroendocrine cells is altered, leading to diarrhea. Because polyalanine expansions within the ARX protein are the most common mutations found in ARX-related disorders, we sought to characterize the enteroendocrine population in human tissue of an ARX(GGC)7 mutation and in a mouse model of the corresponding polyalanine expansion (Arx(GCG)7).
Methods:
Immunohistochemistry and quantitative real-time polymerase chain reaction were the primary modalities used to characterize the enteroendocrine populations. Daily weights were determined for the growth curves, and Oil-Red-O staining on stool and tissue identified neutral fats.
Results:
An expansion of 7 alanines in the first polyalanine tract of both human ARX and mouse Arx altered enteroendocrine differentiation. In human tissue, cholecystokinin, glucagon-like peptide 1, and somatostatin populations were reduced, whereas the chromogranin A population was unchanged. In the mouse model, cholecystokinin and glucagon-like peptide 1 populations were also lost, although the somatostatin-expressing population was increased. The ARX(GGC)7 protein was present in human tissue, whereas the Arx(GCG)7 protein was degraded in the mouse intestine.
Conclusions:
ARX/Arx is required for the specification of a subset of enteroendocrine cells in both humans and mice. Owing to protein degradation, the Arx(GCG)7 mouse recapitulates findings of the intestinal Arx null model, but is not able to further the study of the differential effects of the ARX(GCG)7 protein on its transcriptional targets in the intestine.
doi:10.1097/MPG.0000000000000542
PMCID: PMC4308495  PMID: 25171319
Arx; enteroendocrine dysgenesis; polyalanine
8.  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 retards cell division 
PathoGenetics  2010;3:1.
Background
Aristaless related homeobox (ARX) is a paired-type homeobox gene. ARX function is frequently affected by naturally occurring mutations. Nonsense mutations, polyalanine tract expansions and missense mutations in ARX cause a range of intellectual disability and epilepsy phenotypes with or without additional features including hand dystonia, lissencephaly, autism or dysarthria. Severe malformation phenotypes, such as X-linked lissencephaly with ambiguous genitalia (XLAG), are frequently observed in individuals with protein truncating or missense mutations clustered in the highly conserved paired-type homeodomain.
Results
We have identified two novel point mutations in the R379 residue of the ARX homeodomain; c.1135C>A, p.R379S in a patient with infantile spasms and intellectual disability and c.1136G>T, p.R379L in a patient with XLAG. We investigated these and other missense mutations (R332P, R332H, R332C, T333N: associated with XLAG and Proud syndrome) predicted to affect the nuclear localisation sequences (NLS) flanking either end of the ARX homeodomain. The NLS regions are required for correct nuclear import facilitated by Importin 13 (IPO13). We demonstrate that missense mutations in either the N- or C-terminal NLS regions of the homeodomain cause significant disruption to nuclear localisation of the ARX protein in vitro. Surprisingly, none of these mutations abolished the binding of ARX to IPO13. This was confirmed by co-immunoprecipitation and immmuno fluorescence studies. Instead, tagged and endogenous IPO13 remained bound to the mutant ARX proteins, even in the RanGTP rich nuclear environment. We also identify the microtubule protein TUBA1A as a novel interacting protein for ARX and show cells expressing mutant ARX protein accumulate in mitosis, indicating normal cell division may be disrupted.
Conclusions
We show that the most likely, common pathogenic mechanism of the missense mutations in NLS regions of the ARX homeodomain is inadequate accumulation and distribution of the ARX transcription factor within the nucleus due to sequestration of ARX with IPO13.
doi:10.1186/1755-8417-3-1
PMCID: PMC2819251  PMID: 20148114
9.  Neonatal Estradiol Stimulation Prevents Epilepsy in Arx Model of X-Linked Infantile Spasms Syndrome 
Science translational medicine  2014;6(220):220ra12.
Infantile spasms are a catastrophic form of pediatric epilepsy with inadequate treatment. In patients, mutation of ARX, a transcription factor selectively expressed in neuronal precursors and adult inhibitory interneurons, impairs cell migration and causes a major inherited subtype of the disease X-linked infantile spasms syndrome. Using an animal model, the Arx(GCG)10+7 mouse, we determined that brief estradiol (E2) administration during early postnatal development prevented spasms in infancy and seizures in adult mutants. E2 was ineffective when delivered after puberty or 30 days after birth. Early E2 treatment altered mRNA levels of three downstream targets of Arx (Shox2, Ebf3, and Lgi1) and restored depleted interneuron populations without increasing GABAergic synaptic density. Postnatal E2 treatment may induce lasting transcriptional changes that lead to enduring disease modification and could potentially serve as a therapy for inherited interneuronopathies.
doi:10.1126/scitranslmed.3007231
PMCID: PMC4034383  PMID: 24452264
10.  High-Throughput Analysis of Promoter Occupancy Reveals New Targets for Arx, a Gene Mutated in Mental Retardation and Interneuronopathies 
PLoS ONE  2011;6(9):e25181.
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.
doi:10.1371/journal.pone.0025181
PMCID: PMC3178625  PMID: 21966449
11.  Identification of Arx targets unveils new candidates for controlling cortical interneuron migration and differentiation 
Mutations in the homeobox transcription factor ARX have been found to be responsible for a wide spectrum of disorders extending from phenotypes with severe neuronal migration defects, such as lissencephaly, to mild forms of intellectual disabilities without apparent brain abnormalities, but with associated features of dystonia and epilepsy. Arx expression is mainly restricted to populations of GABA-containing neurons. Studies of the effects of ARX loss of function, either in humans or mutant mice, revealed varying defects, suggesting multiple roles of this gene in brain patterning, neuronal proliferation and migration, cell maturation and differentiation, as well as axonal outgrowth and connectivity. However, to date, little is known about how Arx functions as a transcription factor or which genes it binds and regulates. Recently, we combined chromatin immunoprecipitation and mRNA expression with microarray analysis and identified approximately 1000 gene promoters bound by Arx in transfected neuroblastoma N2a cells and mouse embryonic brain. To narrow the analysis of Arx targets to those most likely to control cortical interneuron migration and/or differentiation, we compare here our data to previously published studies searching for genes enriched or down-regulated in cortical interneurons between E13.5 and E15.5. We thus identified 14 Arx-target genes enriched (Cxcr7, Meis1, Ppap2a, Slc 12a5, Ets2, Phlda1, Egr1, Igf1, Lmo3, Sema6, Lgi1, Alk, Tgfb3, and Napb) and 5 genes specifically down-regulated (Hmgn3, Lmo1, Ebf3, Rasgef1b, and Slit2) in cortical migrating neurons. In this review, we present these genes and discuss how their possible regulation by Arx may lead to the dysfunction of GABAergic neurons, resulting in mental retardation and epilepsy.
doi:10.3389/fncel.2011.00028
PMCID: PMC3280452  PMID: 22355284
ARX; GABA; epilepsy; interneurons; neuronal migration; basal ganglia
12.  Distinct DNA binding and transcriptional repression characteristics related to different ARX mutations 
Neurogenetics  2012;13(1):23-29.
Mutations in the Aristaless-related homeobox gene (ARX) are associated with a wide variety of neurologic disorders including lissencephaly, hydrocephaly, West syndrome, Partington syndrome, and X-linked intellectual disability with or without epilepsy. A genotype-phenotype correlation exists for ARX mutations, however the molecular basis for this association has not been investigated. To begin understanding the molecular basis for ARX mutations, we tested the DNA binding sequence preference and transcriptional repression activity for Arx, deletion mutants and mutants associated with various neurologic disorders. We found DNA binding preferences of Arx are influenced by the amino acid sequences adjacent to the homeodomain. Mutations in the homeodomain show a loss DNA binding activity, while the T333N and P353R homeodomain mutants still possess DNA binding activities, although less than wild type. Transcription repression activity, the primary function of ARX, is reduced in all mutants except the L343Q, which has no DNA binding activity and does not functionally repress Arx targets. These data indicate that mutations in the homeodomain result in not only a loss of DNA binding activity but also loss of transcriptional repression activity. Our results provide novel insights into the pathogenesis of ARX related disorders and possible directions to pursue potential therapeutic interventions.
doi:10.1007/s10048-011-0304-7
PMCID: PMC3279587  PMID: 22252899
ARX; lissencephaly; X-linked intellectual disability and Homeodomain
13.  Infantile Spasms: A Critical Review of Emerging Animal Models 
Epilepsy Currents  2009;9(3):75-81.
Infantile spasms is a developmental epilepsy syndrome with unique clinical and EEG features, a specific pattern of pharmacological responsiveness, and poor outcome in terms of cognition and epilepsy. Despite the devastating nature of infantile spasms, little is known about its pathogenesis. Until recently, there has been no animal model available to investigate the pathophysiology of the syndrome or to generate and test novel therapies. Now, several promising animal models have emerged, spanning the etiological spectrum from genetic causes (e.g., Down syndrome or Aristaless-related homeobox [ARX] mutation) to acquired causes (e.g., endogenous and exogenous toxins or stress hormones with convulsant activity or blockade of neural activity). These new models are discussed in this review, with emphasis on the insights each can provide for understanding, treating, and preventing infantile spasms.
doi:10.1111/j.1535-7511.2009.01299.x
PMCID: PMC2685885  PMID: 19471616
14.  Basic mechanisms of catastrophic epilepsy – overview from animal models 
Brain & development  2013;35(8):748-756.
Infantile spasms are age-specific seizures of infantile epileptic encephalopathies that are usually associated with poor epilepsy and neurodevelopmental outcomes. The current treatments are not always effective and may be associated with significant side effects. Various mechanisms have been proposed as pathogenic for infantile spasms, including cortical or brainstem dysfunction, disruption of normal cortical-subcortical communications, genetic defects, inflammation, stress, developmental abnormalities. Many of these have been recently tested experimentally, resulting into the emergence of several animal models of infantile spasms. The stress theory of spasms yielded the corticotropin releasing hormone (CRH) induced model, which showed the higher proconvulsant potency of CRH in developing rats, although only limbic seizures were observed. Models of acute induction of infantile spasms in rodents include the N-methyl-D-aspartate (NMDA) model of emprosthotonic seizures, the prenatal betamethasone and prenatal stress variants of the NMDA model, and the γ-butyrolactone induced spasms in a Down’s syndrome mouse model. Chronic rodent models of infantile spasms include the tetrodotoxin model and the multiple-hit models in rats, as well as two genetic mouse models of interneuronopathies with infantile spasms due to loss of function of the aristaless X-linked homeobox related gene (ARX). This review discusses the emerging mechanisms for generation of infantile spasms and their associated chronic epileptic and dyscognitive phenotype as well as the recent progress in identifying pathways to better treat this epileptic encephalopathy.
doi:10.1016/j.braindev.2012.12.005
PMCID: PMC3644363  PMID: 23312951
Infantile spasms; mTOR; rapamycin; vigabatrin; carisbamate; ACTH; cognitive; ARX; NMDA
15.  CDKL5 and ARX mutations in males with early-onset epilepsy 
Pediatric neurology  2013;48(5):367-377.
Mutations in CDKL5 and ARX are known causes of early-onset epilepsy and severe developmental delay in males and females. While numerous males with ARX mutations associated with various phenotypes have been reported in the literature, the majority of CDKL5 mutations have been identified in females with a phenotype characterized by early-onset epilepsy, severe global developmental delay, absent speech, and stereotypic hand movements. To date, only ten males with CDKL5 mutations have been reported. Our retrospective study reports on the clinical, neuroimaging and molecular findings of 18 males with early-onset epilepsy caused by either CDKL5 or ARX mutations. The 18 patients include eight new males with CDKL5 mutations and ten with ARX mutations identified through sequence analysis of 266 and 346 males, respectively, at our molecular diagnostic laboratory. Our large data set therefore expands on the number of reported males with CDKL5 mutations and highlights that aberrations of CDKL5 and ARX combined are an important consideration in the genetic forms of early-onset epilepsy.
doi:10.1016/j.pediatrneurol.2012.12.030
PMCID: PMC3742321  PMID: 23583054
16.  CDKL5/STK9 is mutated in Rett syndrome variant with infantile spasms 
Journal of Medical Genetics  2005;42(2):103-107.
Background: Rett syndrome is a severe neurodevelopmental disorder, almost exclusively affecting females and characterised by a wide spectrum of clinical manifestations. Both the classic form and preserved speech variant of Rett syndrome are due to mutations in the MECP2 gene. Several other variants of Rett syndrome have been described. In 1985, Hanefeld described a variant with the early appearance of convulsions. In this variant, the normal perinatal period is soon followed by the appearance of seizures, usually infantile spasms. We have observed two patients with signs of Rett syndrome showing acquired microcephaly and stereotypic midline hand movements. The disease started with generalised convulsions and myoclonic fits at 1.5 months in the first patient and with spasms at 10 days in the other, suggesting a diagnosis of the Hanefeld variant. In these patients, MECP2 point mutations and gross rearrangements were excluded by denaturing high performance liquid chromatography and real time quantitative PCR. The ARX and CDKL5 genes have been associated with West syndrome (infantile spasms, hypsarrhythmia, and mental retardation).
Methods: Based on the clinical overlap between the Hanefeld variant and West syndrome, we analysed ARX and CDKL5 in the two girls.
Results: We found frameshift deletions in CDKL5 in both patients; one in exon 5 (c.163_166delGAAA) and the other in exon 18 (c.2635_2636delCT). CDKL5 was then analysed in 19 classic Rett and 15 preserved speech variant patients, all MECP2 negative, but no mutations were found.
Conclusion: Our results show that CDKL5 is responsible for a rare variant of Rett syndrome characterised by early development of convulsions, usually of the spasm type.
doi:10.1136/jmg.2004.026237
PMCID: PMC1735977  PMID: 15689447
17.  The c.429_452 duplication of the ARX gene: a unique developmental-model of limb kinetic apraxia 
Background
The c.429_452dup24 of the ARX gene is a rare genetic anomaly, leading to X-Linked Intellectual Disability without brain malformation. While in certain cases c.429_452dup24 has been associated with specific clinical patterns such as Partington syndrome, the consequence of this mutation has been also often classified as “non-specific Intellectual Disability”. The present work aims at a more precise description of the clinical features linked to the c.429_452dup24 mutation.
Methods
We clinically reviewed all affected patients identified in France over a five-year period, i.e. 27 patients from 12 different families. Detailed cognitive, behavioural, and motor evaluation, as well as standardized videotaped assessments of oro-lingual and gestural praxis, were performed. In a sub-group of 13 ARX patients, kinematic and MRI studies were further accomplished to better characterize the motor impairment prevalent in the ARX patients group. To ensure that data were specific to the ARX gene mutation and did not result from low-cognitive functioning per se, a group of 27 age- and IQ-matched Down syndrome patients served as control.
Results
Neuropsychological and motor assessment indicated that the c.429_452dup24 mutation constitutes a recognizable clinical syndrome: ARX patients exhibiting Intellectual Disability, without primary motor impairment, but with a very specific upper limb distal motor apraxia associated with a pathognomonic hand-grip. Patients affected with the so-called Partington syndrome, which involves major hand dystonia and orolingual apraxia, exhibit the most severe symptoms of the disorder. The particular “reach and grip” impairment which was observed in all ARX patients, but not in Down syndrome patients, was further characterized by the kinematic data: (i) loss of preference for the index finger when gripping an object, (ii) major impairment of fourth finger deftness, and (iii) a lack of pronation movements. This lack of distal movement coordination exhibited by ARX patients is associated with the loss of independent digital dexterity and is similar to the distortion of individual finger movements and posture observed in Limb Kinetic Apraxia.
Conclusion
These findings suggest that the ARX c.429_452dup24 mutation may be a developmental model for Limb Kinetic Apraxia.
doi:10.1186/1750-1172-9-25
PMCID: PMC4016261  PMID: 24528893
ARX gene mutation; Kinematic study; Limb-kinetic apraxia; X-linked intellectual disability; Partington syndrome
18.  Arx is required for normal enteroendocrine cell development in mice and humans 
Developmental Biology  2012;365(1):175-188.
Enteroendocrine cells of the gastrointestinal (GI) tract play a central role in metabolism, digestion, satiety and lipid absorption, yet their development remains poorly understood. Here we show that Arx, a homeodomain-containing transcription factor, is required for the normal development of mouse and human enteroendocrine cells. Arx expression is detected in a subset of Neurogenin3 (Ngn3)-positive endocrine progenitors and is also found in a subset of hormone-producing cells. In mice, removal of Arx from the developing endoderm results in a decrease of enteroendocrine cell types including gastrin-, glucagon/GLP-1-, CCK-, secretin-producing cell populations and an increase of somatostatin-expressing cells. This phenotype is also observed in mice with endocrine-progenitor-specific Arx ablation suggesting that Arx is required in the progenitor for enteroendocrine cell development. In addition, depletion of human ARX in developing human intestinal tissue results in a profound deficit in expression of the enteroendocrine cell markers CCK, secretin and glucagon while expression of a pan-intestinal epithelial marker, CDX2, and other non-endocrine markers remained unchanged. Taken together, our findings uncover a novel and conserved role of Arx in mammalian endocrine cell development and provide a potential cause for the chronic diarrhea seen in both humans and mice carrying Arx mutations.
doi:10.1016/j.ydbio.2012.02.024
PMCID: PMC3322318  PMID: 22387004
Arx; transcription factor; glucagon; somatostatin; gastrin; CCK; secretin; serotonin; enteroendocrine cells; endocrine progenitors; specification; differentiation
19.  Ohtahara syndrome in a family with an ARX protein truncation mutation (c.81C>G/p.Y27X) 
Aristaless-related homeobox (ARX) gene mutations cause a diverse spectrum of disorders of the human brain, including lissencephaly, various forms of epilepsy and non-syndromic mental retardation. We have identified a novel mutation, c.81C>G (p.Y27X), within the ARX gene in a family with two affected male cousins. One of the boys was diagnosed with an early infantile epileptic encephalopathy also known as Ohtahara syndrome, whereas his cousin had been diagnosed with West syndrome (WS). Both patients have normal genitalia and neither have lissencephaly. The ARX mutation identified is predicted to yield a severely truncated protein of only 26 amino acids and can be considered as a null mutation. Somewhat surprisingly, however, it does not yield the X-linked lissencephaly with ambiguous genitalia (XLAG) syndrome. We proposed that the ARX mRNA translation re-initiated at the next AUG codon at position c.121–123 (aa 41) and, thus, partly rescued these patients from XLAG. Our in vitro studies show that this N-terminally truncated ARX protein (p.M41_C562) is detected by western immunoblot in lysates from cells transiently transfected with an ARX over-expression construct containing the c.81C>G mutation. Although these findings widen the spectrum of clinical phenotypes because of mutations in the ARX gene, they also emphasize the molecular pathogenetic effect of individual mutations as well as the effect of genetic background resulting in intrafamilial clinical heterogeneity for these mutations.
doi:10.1038/ejhg.2009.139
PMCID: PMC2987188  PMID: 19738637
Ohtahara syndrome; burst suppression; ARX gene; West syndrome
20.  Is there a Mendelian transmission ratio distortion of the c.429_452dup(24bp) polyalanine tract ARX mutation? 
European Journal of Human Genetics  2012;20(12):1311-1314.
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.
doi:10.1038/ejhg.2012.61
PMCID: PMC3499743  PMID: 22490986
intellectual disability; polyalanine tract expansions; Mendelian transmission; ARX; meiotic drive
21.  MEF2C Haploinsufficiency features consistent hyperkinesis, variable epilepsy, and has a role in dorsal and ventral neuronal developmental pathways 
Neurogenetics  2013;14(2):99-111.
MEF2C haploinsufficiency syndrome is an emerging neurodevelopmental disorder associated with intellectual disability, autistic features, epilepsy, and abnormal movements. We report 16 new patients with MEF2C haploinsufficiency, including the oldest reported patient with MEF2C deletion at 5q14.3. We detail the neurobehavioral phenotype, epilepsy, and abnormal movements, and compare our subjects with those previously reported in the literature. We also investigate Mef2c expression in the developing mouse forebrain. A spectrum of neurofunctional deficits emerges, with hyperkinesis a consistent finding. Epilepsy varied from absent to severe, and included intractable myoclonic seizures and infantile spasms. Subjects with partial MEF2C deletion were statistically less likely to have epilepsy. Finally, we confirm that Mef2c is present both in dorsal primary neuroblasts and ventral gamma-aminobutyric acid(GABA)ergic interneurons in the forebrain of the developing mouse. Given interactions with several key neurodevelopmental genes such as ARX, FMR1, MECP2, and TBR1, it appears that MEF2C plays a role in several developmental stages of both dorsal and ventral neuronal cell types.
doi:10.1007/s10048-013-0356-y
PMCID: PMC3773516  PMID: 23389741
MEF2C haploinsufficiency; Intellectual disability; Autism; Infant-onset myoclonic epilepsy; Infantile spasms; Hyperkinesis; Deletion 5q14.3
22.  Distinct Molecular Pathways for Development of Telencephalic Interneuron Subtypes Revealed Through Analysis of Lhx6 Mutants 
Here we analyze the role of the Lhx6 lim-homeobox transcription factor in regulating the development of subsets of neocortical, hippocampal and striatal interneurons. An Lhx6 loss-of-function allele, that expresses placental alkaline phosphatase (PLAP), allowed analysis of the development and fate of Lhx6-expressing interneurons in mice lacking this homeobox transcription factor. There are Lhx6+;Dlx+ and Lhx6−;Dlx+ subtypes of tangentially migrating interneurons. Most interneurons in Lhx6PLAP/PLAP mutants migrate to the cortex, although less efficiently, and exhibit defects in populating the marginal zone and superficial parts of the neocortical plate. By contrast, migration to superficial parts of the hippocampus is not seriously affected. Furthermore, whereas parvalbumin+ and somatostatin+ interneurons do not differentiate, NPY+ interneurons are present; we suggest that these NPY+ interneurons are derived from the Lhx6−;Dlx+ subtype. Striatal interneurons show deficits distinct from pallial interneurons, including a reduction in the NPY+ subtype. We provide evidence that Lhx6 mediates these effects through promoting expression of receptors that regulate interneuron migration (ErbB4, CXCR4, CXCR7), and through promoting the expression of transcription factors either known (Arx) or implicated (bMaf, Cux2, and NPAS1) in controlling interneuron development.
doi:10.1002/cne.21772
PMCID: PMC2547494  PMID: 18613121
Lhx6; Dlx; GABAergic; Interneuron; NPY
23.  Mutations in ARX Result in Several Defects Involving GABAergic Neurons 
Genetic investigations of X-linked mental retardation have demonstrated the implication of ARX in a wide spectrum of disorders extending from phenotypes with 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. These investigations have in recent years directed attention to the role of this gene in brain development. Analysis of its spatio-temporal localization profile revealed expression in telencephalic structures at all stages of development, mainly restricted to populations of GABA-containing neurons. Furthermore, studies of the effects of ARX loss of function either in humans or in lines of mutant mice revealed varying defects, suggesting multiple roles of this gene during development. In particular, Arx has been shown to contribute to almost all fundamental processes of brain development: patterning, neuronal proliferation and migration, cell maturation and differentiation, as well as axonal outgrowth and connectivity. In this review, we will present and discuss recent findings concerning the role of ARX in brain development and how this information will be useful to better understand the pathophysiological mechanisms of mental retardation and epilepsy associated with ARX mutations.
doi:10.3389/fncel.2010.00004
PMCID: PMC2841486  PMID: 20300201
ARX; GABA; lissencephaly; epilepsy; interneurons; neuronal migration; basal ganglia
24.  MRX87 family with Aristaless X dup24bp mutation and implication for polyAlanine expansions 
BMC Medical Genetics  2007;8:25.
Background
Cognitive impairments are heterogeneous conditions, and it is estimated that 10% may be caused by a defect of mental function genes on the X chromosome. One of those genes is Aristaless related homeobox (ARX) encoding a polyA-rich homeobox transcription factor essential for cerebral patterning and its mutations cause different neurologic disorders. We reported on the clinical and genetic analysis of an Italian family with X-linked mental retardation (XLMR) and intra-familial heterogeneity, and provided insight into its molecular defect.
Methods
We carried out on linkage-candidate gene studies in a new MRX family (MRX87). All coding regions and exon-intron boundaries of ARX gene were analysed by direct sequencing.
Results
MRX87 patients had moderate to profound cognition impairment and a combination of minor congenital anomalies. The disease locus, MRX87, was mapped between DXS7104 and DXS1214, placing it in Xp22-p21 interval, a hot spot region for mental handicap. An in frame duplication of 24 bp (ARXdup24) in the second polyAlanine tract (polyA_II) in ARX was identified.
Conclusion
Our study underlines the role of ARXdup24 as a critical mutational site causing mental retardation linked to Xp22. Phenotypic heterogeneity of MRX87 patients represents a new observation relevant to the functional consequences of polyAlanine expansions enriching the puzzling complexity of ARXdup24-linked diseases.
doi:10.1186/1471-2350-8-25
PMCID: PMC1868705  PMID: 17480217
25.  CDKL5 mutations cause infantile spasms, early onset seizures, and severe mental retardation in female patients 
Journal of Medical Genetics  2006;43(9):729-734.
Objective
To determine the frequency of mutations in CDKL5 in both male and female patients with infantile spasms or early onset epilepsy of unknown cause, and to consider whether the breadth of the reported phenotype would be extended by studying a different patient group.
Methods
Two groups of patients were investigated for CDKL5 mutations. Group 1 comprised 73 patients (57 female, 16 male) referred to Cardiff for CDKL5 analysis, of whom 49 (42 female, 7 male) had epileptic seizure onset in the first six months of life. Group 2 comprised 26 patients (11 female, 15 male) with infantile spasms previously recruited to a clinical trial, the UK Infantile Spasms Study. Where a likely pathogenic mutation was identified, further clinical data were reviewed.
Results
Seven likely pathogenic mutations were found among female patients from group 1 with epileptic seizure onset in the first six months of life, accounting for seven of the 42 in this group (17%). No mutations other than the already published mutation were found in female patients from group 2, or in any male patient from either study group. All patients with mutations had early signs of developmental delay and most had made little developmental progress. Further clinical information was available for six patients: autistic features and tactile hypersensitivity were common but only one had suggestive Rett‐like features. All had a severe epileptic seizure disorder, all but one of whom had myoclonic jerks. The EEG showed focal or generalised changes and in those with infantile spasms, hypsarrhythmia. Slow frequencies were seen frequently with a frontal or fronto‐temporal predominance and high amplitudes.
Conclusions
The spectrum of the epileptic seizure disorder, and associated EEG changes, in those with CDKL5 mutations is broader than previously reported. CDKL5 mutations are a significant cause of infantile spasms and early epileptic seizures in female patients, and of a later intractable seizure disorder, irrespective of whether they have suspected Rett syndrome. Analysis should be considered in these patients in the clinical setting.
doi:10.1136/jmg.2006.041467
PMCID: PMC2564572  PMID: 16611748
CDKL5 ; infantile spasms; epilepsy; Rett syndrome

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