Refining the phenotype associated with GNB1 mutations: Clinical data on 18 newly identified patients and review of the literature.

De novo germline mutations in GNB1 have been associated with a neurodevelopmental phenotype. To date, 28 patients with variants classified as pathogenic have been reported. We add 18 patients with de novo mutations to this cohort, including a patient with mosaicism for a GNB1 mutation who presented with a milder phenotype. Consistent with previous reports, developmental delay in these patients was moderate to severe, and more than half of the patients were non-ambulatory and nonverbal. The most observed substitution affects the p.Ile80 residue encoded in exon 6, with 28% of patients carrying a variant at this residue. Dystonia and growth delay were observed more frequently in patients carrying variants in this residue, suggesting a potential genotype-phenotype correlation. In the new cohort of 18 patients, 50% of males had genitourinary anomalies and 61% of patients had gastrointestinal anomalies, suggesting a possible association of these findings with variants in GNB1. In addition, cutaneous mastocytosis, reported once before in a patient with a GNB1 variant, was observed in three additional patients, providing further evidence for an association to GNB1. We will review clinical and molecular data of these new cases and all previously reported cases to further define the phenotype and establish possible genotype-phenotype correlations.

Phenotypic expansion and progression of SPATA7-associated retinitis pigmentosa.

PURPOSE: To report an unusual phenotype of retinitis pigmentosa (RP) caused by compound heterozygous mutations in SPATA7, and describe the progression over a two year follow-up period. METHODS: Retrospective case study. RESULTS: A 63-year-old man with a long history of nyctalopia, progressive visual field constriction, and a recent subacute decrease in visual acuity of the left eye presented for evaluation of a suspected retinal degeneration. Multimodal retinal imaging and functional assessment with full-field electroretinogram suggested a severe rod-cone dysfunction masquerading as a choroideremia-like phenotype. A vitreous opacity was found to explain recent changes in the left eye and a 25-guage vitrectomy and membrane peel was performed, yielding no change in visual acuity. Whole-exome sequencing revealed compound heterozygous variants in SPATA7 that were predicted to be pathogenic. CONCLUSIONS: Compound heterozygous c.1100A > G, p.(Y367C) and c.1102_1103delCT, p.(L368Efs*4) variants in SPATA7 manifest as an unusual RP phenotype in this case, showing extensive choroidal sclerosis and retinal pigment epithelium (RPE) atrophy with evidence of progression over two years on multimodal imaging.

Ena/VASP Enabled is a highly processive actin polymerase tailored to self-assemble parallel-bundled F-actin networks with Fascin.

Filopodia are exploratory finger-like projections composed of multiple long, straight, parallel-bundled actin filaments that protrude from the leading edge of migrating cells. Drosophila melanogaster Enabled (Ena) is a member of the Ena/vasodilator-stimulated phosphoprotein protein family, which facilitates the assembly of filopodial actin filaments that are bundled by Fascin. However, the mechanism by which Ena and Fascin promote the assembly of uniformly thick F-actin bundles that are capable of producing coordinated protrusive forces without buckling is not well understood. We used multicolor evanescent wave fluorescence microscopy imaging to follow individual Ena molecules on both single and Fascin-bundled F-actin in vitro. Individual Ena tetramers increase the elongation rate approximately two- to threefold and inhibit capping protein by remaining processively associated with the barbed end for an average of ∼10 s in solution, for ∼60 s when immobilized on a surface, and for ∼110 s when multiple Ena tetramers are clustered on a surface. Ena also can gather and simultaneously elongate multiple barbed ends. Collectively, these properties could facilitate the recruitment of Fascin and initiate filopodia formation. Remarkably, we found that Ena's actin-assembly properties are tunable on Fascin-bundled filaments, facilitating the formation of filopodia-like F-actin networks without tapered barbed ends. Ena-associated trailing barbed ends in Fascin-bundled actin filaments have approximately twofold more frequent and approximately fivefold longer processive runs, allowing them to catch up with leading barbed ends efficiently. Therefore, Fascin and Ena cooperate to extend and maintain robust filopodia of uniform thickness with aligned barbed ends by a unique mechanistic cycle.

NeuroSCORE is a genome-wide omics-based model that identifies candidate disease genes of the central nervous system.

To identify candidate disease genes of central nervous system (CNS) phenotypes, we created the Neurogenetic Systematic Correlation of Omics-Related Evidence (NeuroSCORE). We identified five genome-wide metrics highly associated with CNS phenotypes to score 19,601 protein-coding genes. Genes scored one point per metric (range: 0-5), identifying 8298 scored genes (scores ≥ 1) and 1601 "high scoring" genes (scores ≥ 3). Using logistic regression, we determined the odds ratio that genes with a NeuroSCORE from 1 to 5 would be associated with known CNS-related phenotypes compared to genes that scored zero. We tested NeuroSCORE using microarray copy number variants (CNVs) in case-control cohorts and aggregate mouse model data. High scoring genes are associated with CNS phenotypes (OR = 5.5, p < 2E-16), enriched in case CNVs, and mouse ortholog genes that cause behavioral and nervous system abnormalities. We identified 1058 high scoring genes with no disease association in OMIM. Transforming the logistic regression results indicates high scoring genes have an 84-92% chance of being associated with a CNS phenotype. Top scoring genes include GRIA1, MAP4K4, SF1, TNPO2, and ZSWIM8. Finally, we interrogated CNVs in the Clinical Genome Resource, finding the majority of clinically significant CNVs contain high scoring genes. These findings can direct future research and improve molecular diagnostics.

Paroxysmal oculogyric dystonia associated with a de novo 3q29 microdeletion.

3q29 deletion syndrome is caused by a heterozygous 1.6 Mb deletion on chromosome 3, which occurs in about 1 in 30 000 births. Phenotypic features of this syndrome include mild-to-moderate intellectual disability, autism spectrum disorder, slightly dysmorphic facial features, ataxic gait, and chest-wall deformity. Gastrointestinal disorders, dental abnormalities, feeding problems during infancy, recurrent ear infections, and heart defects have also been observed. Since the incidence of the deletion is rare, the phenotype has not been fully described, particularly in adults. This report describes a young adult female with 3q29 deletion syndrome, autism spectrum disorder, intellectual disability, and anxiety who experienced a sustained, non-medication induced paroxysmal oculogyric dystonia which responded to anticholinergic and antihistaminic medications. This is the first report of paroxysmal oculogyric dystonia associated with this deletion, possibly expanding the phenotypic features of this microdeletion syndrome.

Abelson kinase acts as a robust, multifunctional scaffold in regulating embryonic morphogenesis.

Abelson family kinases (Abls) are key regulators of cell behavior and the cytoskeleton during development and in leukemia. Abl's SH3, SH2, and tyrosine kinase domains are joined via a linker to an F-actin-binding domain (FABD). Research on Abl's roles in cell culture led to several hypotheses for its mechanism of action: 1) Abl phosphorylates other proteins, modulating their activity, 2) Abl directly regulates the cytoskeleton via its cytoskeletal interaction domains, and/or 3) Abl is a scaffold for a signaling complex. The importance of these roles during normal development remains untested. We tested these mechanistic hypotheses during Drosophila morphogenesis using a series of mutants to examine Abl's many cell biological roles. Strikingly, Abl lacking the FABD fully rescued morphogenesis, cell shape change, actin regulation, and viability, whereas kinase-dead Abl, although reduced in function, retained substantial rescuing ability in some but not all Abl functions. We also tested the function of four conserved motifs in the linker region, revealing a key role for a conserved PXXP motif known to bind Crk and Abi. We propose that Abl acts as a robust multidomain scaffold with different protein motifs and activities contributing differentially to diverse cellular behaviors.

CellGeo: a computational platform for the analysis of shape changes in cells with complex geometries.

Cell biologists increasingly rely on computer-aided image analysis, allowing them to collect precise, unbiased quantitative results. However, despite great progress in image processing and computer vision, current computational approaches fail to address many key aspects of cell behavior, including the cell protrusions that guide cell migration and drive morphogenesis. We developed the open source MATLAB application CellGeo, a user-friendly computational platform to allow simultaneous, automated tracking and analysis of dynamic changes in cell shape, including protrusions ranging from filopodia to lamellipodia. Our method maps an arbitrary cell shape onto a tree graph that, unlike traditional skeletonization algorithms, preserves complex boundary features. CellGeo allows rigorous but flexible definition and accurate automated detection and tracking of geometric features of interest. We demonstrate CellGeo's utility by deriving new insights into (a) the roles of Diaphanous, Enabled, and Capping protein in regulating filopodia and lamellipodia dynamics in Drosophila melanogaster cells and (b) the dynamic properties of growth cones in catecholaminergic a-differentiated neuroblastoma cells.

Enabled negatively regulates diaphanous-driven actin dynamics in vitro and in vivo.

Actin regulators facilitate cell migration by controlling cell protrusion architecture and dynamics. As the behavior of individual actin regulators becomes clear, we must address why cells require multiple regulators with similar functions and how they cooperate to create diverse protrusions. We characterized Diaphanous (Dia) and Enabled (Ena) as a model, using complementary approaches: cell culture, biophysical analysis, and Drosophila morphogenesis. We found that Dia and Ena have distinct biochemical properties that contribute to the different protrusion morphologies each induces. Dia is a more processive, faster elongator, paralleling the long, stable filopodia it induces in vivo, while Ena promotes filopodia with more dynamic changes in number, length, and lifetime. Acting together, Ena and Dia induce protrusions distinct from those induced by either alone, with Ena reducing Dia-driven protrusion length and number. Consistent with this, EnaEVH1 binds Dia directly and inhibits DiaFH1FH2-mediated nucleation in vitro. Finally, Ena rescues hemocyte migration defects caused by activated Dia.