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Correspondenc

Missense variant contribution to USP9X-female syndrome

USP9X is an X-chromosome gene that escapes X-inactivation. Loss or compromised function of USP9X leads to neurodevelopmental disorders in males and females. While males are impacted primarily by hemizygous partial loss-of-function missense variants, in females de novo heterozygous complete loss-of-function mutations predominate, and give rise to the clinically recognisable USP9X-female syndrome. Here we provide evidence of the contribution of USP9X missense and small in-frame deletion variants in USP9X-female syndrome also. We scrutinise the pathogenicity of eleven such variants, ten of which were novel. Combined application of variant prediction algorithms, protein structure modelling, and assessment under clinically relevant guidelines universally support their pathogenicity. The core phenotype of this cohort overlapped with previous descriptions of USP9X-female syndrome, but exposed heightened variability. Aggregate phenotypic information of 35 currently known females with predicted pathogenic variation in USP9X reaffirms the clinically recognisable USP9X-female syndrome, and highlights major differences when compared to USP9X-male associated neurodevelopmental disorders

Seizures Are Regulated by Ubiquitin-specific Peptidase 9 X-linked (USP9X), a De-Ubiquitinase

<div><p>Epilepsy is a common disabling disease with complex, multifactorial genetic and environmental etiology. The small fraction of epilepsies subject to Mendelian inheritance offers key insight into epilepsy disease mechanisms; and pathologies brought on by mutations in a single gene can point the way to generalizable therapeutic strategies. Mutations in the PRICKLE genes can cause seizures in humans, zebrafish, mice, and flies, suggesting the seizure-suppression pathway is evolutionarily conserved. This pathway has never been targeted for novel anti-seizure treatments. Here, the mammalian PRICKLE-interactome was defined, identifying prickle-interacting proteins that localize to synapses and a novel interacting partner, USP9X, a substrate-specific de-ubiquitinase. PRICKLE and USP9X interact through their carboxy-termini; and USP9X de-ubiquitinates PRICKLE, protecting it from proteasomal degradation. In forebrain neurons of mice, USP9X deficiency reduced levels of Prickle2 protein. Genetic analysis suggests the same pathway regulates Prickle-mediated seizures. The seizure phenotype was suppressed in <i>prickle</i> mutant flies by the small-molecule USP9X inhibitor, Degrasyn/WP1130, or by reducing the dose of <i>fat facets</i> a <i>USP9X</i> orthologue. <i>USP9X</i> mutations were identified by resequencing a cohort of patients with epileptic encephalopathy, one patient harbored a <i>de novo</i> missense mutation and another a novel coding mutation. Both <i>USP9X</i> variants were outside the PRICKLE-interacting domain. These findings demonstrate that USP9X inhibition can suppress <i>prickle</i>-mediated seizure activity, and that <i>USP9X</i> variants may predispose to seizures. These studies point to a new target for anti-seizure therapy and illustrate the translational power of studying diseases in species across the evolutionary spectrum.</p></div

PRICKLE interacts with USP9X via its carboxyl terminal.

<p> <b>A.</b> PRICKLE1 and PRICKLE2 interact with endogenous Bcr, Tanc2, and Usp9x in NGF-differentiated PC12 cells. GFP immunoprecipitates from stable lines expressing GFP, GFP-PRICKLE1 or GFP-PRICKLE2 confirm that PRICKLE interacts with Bcr, Usp9x and Tanc2. <b>B</b>. Flag immunoprecipitates, from HEK293T cells overexpressing flag-tagged PRICKLE1 or PRICKLE2, show endogenous USP9X physically interacts with PRICKLE. <b>C, D.</b> Schematic of PRICKLE1(C) and PRICKLE2(D) constructs. Flag-immunoprecipitates from HEK293T cells overexpressing the indicated constructs were analyzed by anti-USP9X Western blotting. Both PRICKLE 1 and 2 interact with USP9X via their C-termini while BCR binding mapped to their N-termini.</p

PRICKLE1 Interaction with SYNAPSIN I Reveals a Role in Autism Spectrum Disorders

<div><p>The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, <i>PRICKLE1</i> missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in <i>Prickle1^+/−</i> mice and <i>Drosophila</i>, yeast, and neuronal cell lines. We show that mice with <i>Prickle1</i> mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the <i>SYN1</i> region mutated in ASD and epilepsy. Finally, a mutation in <i>PRICKLE1</i> disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest <i>PRICKLE1</i> mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.</p></div

Prickle interactome.

<p>We combined findings from our proteomics interaction experiment and public databases to generate a prickle interactome. We used the MetaCore (MetaCore, GeneGO Inc., St. Joseph, MI, USA) networking function and String database 9.1 to curate interaction maps of the proteins identified. Information for identified interactions is obtained from several sources including but not limited to genomic context, database imports (PPI and pathway databases), high-throughput experiments, co-expression, and text mining. We uploaded our lists of proteins from LC-MS/MS into the software programs and exported the networks into Cytoscape 2.7.0 for manipulation of the network appearance. (Nodes, circles; Edges, lines). Red lines correspond to interactions observed by our labs using yeast-2-hybrid and IP-MS approaches. The extended interactome was generated as we have previously described. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005022#pgen.1005022.ref001" target="_blank">1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005022#pgen.1005022.ref006" target="_blank">6</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005022#pgen.1005022.ref009" target="_blank">9</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005022#pgen.1005022.ref018" target="_blank">18</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005022#pgen.1005022.ref019" target="_blank">19</a>] Prickle1 and Prickle2 interact with known synaptic proteins. The interaction with USP9X is novel.</p