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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., Competing Interests: The authors have declared that no competing interests exist.

The systematic screening of Rett syndrome (RTT) patients for pathogenetic sequence variations has focused on three genes that have been associated with RTT or related clinical phenotypes, namely MECP2, CDKL5, and FOXG1. More recently, it has been suggested that phenotypes associated with TCF4 and MEF2C mutations may represent a form of RTT. Here we report on the screening of the TCF4 and MEF2C genes in a cohort of 81 classical, atypical, and incomplete atypical RTT patients harboring no known mutations in MECP2, CDKL5, and FOXG1 genes. No pathogenetic sequence variations were identified in the MEF2C gene in our cohort. However, a frameshift mutation in TCF4 was identified in a patient with a clinical diagnosis of "variant" RTT, in whom the clinical evolution later raised the possibility of Pitt-Hopkins syndrome. Although our results suggest that these genes are not commonly associated with RTT, we note the clinical similarity between RTT and Pitt-Hopkins syndrome, and suggest that RTT patients with no mutation identified in MECP2 be considered for molecular screening of the TCF4 gene., (Copyright © 2012 Wiley Periodicals, Inc.)

Rett syndrome (RTT) is a severe neurodevelopmental disorder affecting females almost exclusively and is characterized by a wide spectrum of clinical manifestations. Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene have been found in up to 95% of classical RTT cases and a lesser proportion of atypical cases. Recently, mutations in another X-linked gene, CDKL5 (cyclin-dependent kinase-like 5) have been found to cause atypical RTT, in particular the early onset seizure (Hanefeld variant) and one female with autism. In this study we screened several cohorts of children for CDKL5 mutations, totaling 316 patients, including individuals with a clinical diagnosis of RTT but who were negative for MECP2 mutations (n=102), males with X-linked mental retardation (n=9), patients with West syndrome (n=52), patients with autism (n=59), patients with epileptic encephalopathy (n=33), patients with Aicardi syndrome (n=7) and other patients with intellectual disability with or without seizures (n=54). In all, seven polymorphic variations and four de novo mutations (c.586C>T [p.S196L]; c.58G>C [p.G20R]; c.2504delC [p.P835fs]; deletion of exons 1-3) were identified, and in all instances of the latter the clinical phenotype was that of an epileptic encephalopathy. These results suggest that pathogenic CDKL5 mutations are unlikely to be identified in the absence of severe early-onset seizures and highlight the importance of screening for large intragenic and whole gene deletions.

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.

Comprehensive genetic screening programs have led to the identification of pathogenic methyl-CpG-binding protein 2 (MECP2) mutations in up to 95% of classical Rett syndrome (RTT) patients. This high rate of mutation detection can partly be attributed to specialised techniques that have enabled the detection of large deletions in a substantial fraction of otherwise mutation-negative patients. These cases would normally be missed by the routine PCR-based screening strategies. Here, we have identified large multi-exonic deletions in 12/149 apparently mutation-negative RTT patients using multiplex ligation-dependent probe amplification (MLPA). These deletions were subsequently characterised using real-time quantitative PCR (qPCR) and long-range PCR with the ultimate aim of defining the exact nucleotide positions of the breakpoints and rearrangements. We detected an apparent deletion in one further patient using MLPA; however, this finding was contradicted by subsequent qPCR and long-range PCR results. The patient group includes an affected brother and sister with a large MECP2 deletion also present in their carrier mother. The X chromosome inactivation pattern of all female patients in this study was determined, which, coupled with detailed clinical information, allowed meaningful genotype-phenotype correlations to be drawn. This study reaffirms the view that large MECP2 deletions are an important cause of both classical and atypical RTT syndrome, and cautions that apparent deletions detected using high-throughput diagnostic techniques require further characterisation.

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

The Aristaless-related homeobox gene, ARX, is an important transcription factor with a crucial role in forebrain, pancreas and testes development. At least fifty-nine mutations have been described in the ARX gene in seven X-chromosome linked disorders involving mental retardation. Recent studies with ARX screening suggest that the gene is mutated in 9.5% of X-linked families with these disorders. Two different polyalanine expansion mutations represent 46% of all currently known mutations and show considerable pleiotropy. The ARX gene is emerging as one of the more important disease-causing genes on the X chromosome and ought to be considered for routine screening. Although the normal Arx protein is known to be a bifunctional transcriptional activator and repressor, the complete biochemical characterization of the normal and mutated ARX awaits further investigation. Pax4 was identified as one of the ARX target genes, and both proteins have crucial functions in endocrine mouse pancreas alpha-cell and beta-cell lineage specification.