To determine the phenotypic spectrum caused by mutations in GRIN1 encoding the NMDA receptor subunit GluN1 and to investigate their underlying functional pathophysiology.

Selective pericyte loss, the histological hallmark of early diabetic retinopathy (DR), enhances the breakdown of the blood-retinal barrier (BRB) in diabetes. However, the role of pericytes on BRB alteration in diabetes and the signaling pathways involved in their effects are currently unknown. To understand the role of diabetes-induced molecular alteration of pericytes, we performed transcriptomic analysis of sorted retinal pericytes from mice model of diabetes. Retinal tissue from non-diabetic and diabetic (duration 3 months) mouse eyes (n = 10 in each group) were used to isolate pericytes through fluorescent activated cell sorting (FACS) using pericyte specific fluorescent antibodies, PDGFRb-APC. For RNA sequencing and qPCR analysis, a cDNA library was generated using template switching oligo and the resulting libraries were sequenced using paired-end Illumina sequencing. Molecular functional pathways were analyzed using differentially expressed genes (DEGs). Differential expression analysis revealed 217 genes significantly upregulated and 495 genes downregulated, in pericytes isolated from diabetic animals. These analyses revealed a core set of differentially expressed genes that could potentially contribute to the pericyte dysfunction in diabetes and highlighted the pattern of functional connectivity between key candidate genes and blood retinal barrier alteration mechanisms. The top up-regulated gene list included: Ext2, B3gat3, Gpc6, Pip5k1c and Pten and down-regulated genes included: Notch3, Xbp1, Gpc4, Atp1a2 and AKT3. Out of these genes, we further validated one of the down regulated genes, Notch 3 and its role in BRB alteration in diabetic retinopathy. We confirmed the downregulation of Notch3 expression in human retinal pericytes exposed to Advanced Glycation End-products (AGEs) treatment mimicking the chronic hyperglycemia effect. Exploration of pericyte-conditioned media demonstrated that loss of NOTCH3 in pericyte led to increased permeability of endothelial cell monolayers. Collectively, we identify a role for NOTCH3 in pericyte dysfunction in diabetes. Further validation of other DEGs to identify cell specific molecular change through whole transcriptomic approach in diabetic retina will provide novel insight into the pathogenesis of DR and novel therapeutic targets.

The human RNA helicase DDX6 is an essential component of membrane-less organelles called processing bodies (PBs). PBs are involved in mRNA metabolic processes including translational repression via coordinated storage of mRNAs. Previous studies in human cell lines have implicated altered DDX6 in molecular and cellular dysfunction, but clinical consequences and pathogenesis in humans have yet to be described. Here, we report the identification of five rare de novo missense variants in DDX6 in probands presenting with intellectual disability, developmental delay, and similar dysmorphic features including telecanthus, epicanthus, arched eyebrows, and low-set ears. All five missense variants (p.His372Arg, p.Arg373Gln, p.Cys390Arg, p.Thr391Ile, and p.Thr391Pro) are located in two conserved motifs of the RecA-2 domain of DDX6 involved in RNA binding, helicase activity, and protein-partner binding. We use functional studies to demonstrate that the first variants identified (p.Arg373Gln and p.Cys390Arg) cause significant defects in PB assembly in primary fibroblast and model human cell lines. These variants' interactions with several protein partners were also disrupted in immunoprecipitation assays. Further investigation via complementation assays included the additional variants p.Thr391Ile and p.Thr391Pro, both of which, similarly to p.Arg373Gln and p.Cys390Arg, demonstrated significant defects in P-body assembly. Complementing these molecular findings, modeling of the variants on solved protein structures showed distinct spatial clustering near known protein binding regions. Collectively, our clinical and molecular data describe a neurodevelopmental syndrome associated with pathogenic missense variants in DDX6. Additionally, we suggest DDX6 join the DExD/H-box genes DDX3X and DHX30 in an emerging class of neurodevelopmental disorders involving RNA helicases.

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TRPM3 is a temperature- and neurosteroid-sensitive plasma membrane cation channel expressed in a variety of neuronal and non-neuronal cells. Recently, rare de novo variants in TRPM3 were identified in individuals with developmental and epileptic encephalopathy, but the link between TRPM3 activity and neuronal disease remains poorly understood. We previously reported that two disease-associated variants in TRPM3 lead to a gain of channel function . Here, we report a further 10 patients carrying one of seven additional heterozygous TRPM3 missense variants. These patients present with a broad spectrum of neurodevelopmental symptoms, including global developmental delay, intellectual disability, epilepsy, musculo-skeletal anomalies, and altered pain perception. We describe a cerebellar phenotype with ataxia or severe hypotonia, nystagmus, and cerebellar atrophy in more than half of the patients. All disease-associated variants exhibited a robust gain-of-function phenotype, characterized by increased basal activity leading to cellular calcium overload and by enhanced responses to the neurosteroid ligand pregnenolone sulfate when co-expressed with wild-type TRPM3 in mammalian cells. The antiseizure medication primidone, a known TRPM3 antagonist, reduced the increased basal activity of all mutant channels. These findings establish gain-of-function of TRPM3 as the cause of a spectrum of autosomal dominant neurodevelopmental disorders with frequent cerebellar involvement in humans and provide support for the evaluation of TRPM3 antagonists as a potential therapy.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutation in the X-linked MECP2 gene, encoding methyl-CpG-binding protein 2. We have created a mouse model ( Mecp2 A140V "knock-in" mutant) expressing the recurrent human MECP2 A140V mutation linked to an X-linked mental retardation/Rett syndrome phenotype. Morphological analyses focused on quantifying soma and nucleus size were performed on primary hippocampus and cerebellum granule neuron (CGN) cultures from mutant ( Mecp2A140V/y) and wild type ( Mecp2+/y) male mice. Cultured hippocampus and cerebellar granule neurons from mutant animals were significantly smaller than neurons from wild type animals. We also examined soma size in hippocampus neurons from individual female transgenic mice that express both a mutant  (maternal allele) and a wild type Mecp2 gene linked to an eGFP transgene (paternal allele). In cultures from such doubly heterozygous female mice, the size of neurons expressing the mutant (A140V) allele also showed a significant reduction compared to neurons expressing wild type MeCP2, supporting a cell-autonomous role for MeCP2 in neuronal development. IGF-1 (insulin growth factor-1) treatment of neuronal cells from Mecp2 mutant mice rescued the soma size phenotype. We also found that Mecp2  mutation leads to down-regulation of the mTOR signaling pathway, known to be involved in neuronal size regulation. Our results suggest that i) reduced neuronal size is an important in vitro cellular phenotype of Mecp2 mutation in mice, and ii) MeCP2 might play a critical role in the maintenance of neuronal structure by modulation of the mTOR pathway. The definition of a quantifiable cellular phenotype supports using neuronal size as a biomarker in the development of a high-throughput, in vitro assay to screen for compounds that rescue small neuronal phenotype ("phenotypic assay").

Mutations disrupting presynaptic protein TBC1D24 are associated with a variable neurological phenotype, including DOORS syndrome, myoclonic epilepsy, early-infantile epileptic encephalopathy, and non-syndromic hearing loss. In this report, we describe a family segregating autosomal dominant epilepsy, and a 37-year-old Caucasian female with a severe neurological phenotype including epilepsy, Parkinsonism, psychosis, visual and auditory hallucinations, gait ataxia and intellectual disability. Whole exome sequencing revealed two missense mutations in the TBC1D24 gene segregating within this family (c.1078C>T; p.Arg360Cys and c.404C>T; p.Pro135Leu). The female proband who presents with a severe neurological phenotype carries both of these mutations in a compound heterozygous state. The p.Pro135Leu variant, however, is present in the proband's mother and sibling as well, and is consistent with an autosomal dominant pattern linked to tonic-clonic and myoclonic epilepsy. In conclusion, we describe a single family in which TBC1D24 mutations cause expanded dominant and recessive phenotypes. In addition, we discuss and highlight that some variants in TBC1D24 might cause a dominant susceptibility to epilepsy.

Recently, mutations in the zinc finger MYND-type containing 11 (ZMYND11) gene were identified in patients with autism spectrum disorders, intellectual disability, aggression, and complex neuropsychiatric features, supporting that this gene is implicated in 10p15.3 microdeletion syndrome. We report a novel de novo variant in the ZMYND11 gene (p.Ser421Asn) in a patient with a complex neurodevelopmental phenotype. The patient is a 24-yr-old Caucasian/Filipino female with seizures, global developmental delay, sensorineural hearing loss, hypotonia, dysmorphic features, and other features including a happy disposition and ataxic gait similar to Angelman syndrome. In addition, this patient had uncommon features including eosinophilic esophagitis and multiple, severe allergies not described in similar ZMYND11 cases. This new case further supports the association of ZMYND11 with autistic-like phenotypes and suggests that ZMYND11 should be included in the list of potentially causative candidate genes in cases with complex neurodevelopmental phenotypes.

The proteins encoded by TELO2, TTI1, and TTI2 interact to form the TTT complex, a co-chaperone for maturation of the phosphatidylinositol 3-kinase-related protein kinases (PIKKs). Here we report six affected individuals from four families with intellectual disability (ID) and neurological and other congenital abnormalities associated with compound heterozygous variants in TELO2. Although their fibroblasts showed reduced steady-state levels of TELO2 and the other components of the TTT complex, PIKK functions were normal in cellular assays. Our results suggest that these TELO2 missense variants result in loss of function, perturb TTT complex stability, and cause an autosomal-recessive syndromic form of ID.

The controlled recruitment of monocytes from the circulation to the site of injury and their differentiation into tissue macrophages are critical events in the reconstitution of tissue integrity. Subsets of monocytes/macrophages have been implicated in the pathogenesis of atherosclerosis and tumor vascularity; however, the significance of monocyte heterogeneity in physiologic neovascularization is just emerging.

Description of a new variant of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) gene causing congenital myasthenic syndrome (CMS) in 3 children from 2 unrelated families.

The robust detection of disease-associated splice events from RNAseq data is challenging due to the potential confounding effect of gene expression levels and the often limited number of patients with relevant RNAseq data. Here we present a novel statistical approach to splicing outlier detection and differential splicing analysis. Our approach tests for differences in the percentages of sequence reads representing local splice events. We describe a software package called Bisbee which can predict the protein-level effect of splice alterations, a key feature lacking in many other splicing analysis resources. We leverage Bisbee's prediction of protein level effects as a benchmark of its capabilities using matched sets of RNAseq and mass spectrometry data from normal tissues. Bisbee exhibits improved sensitivity and specificity over existing approaches and can be used to identify tissue-specific splice variants whose protein-level expression can be confirmed by mass spectrometry. We also applied Bisbee to assess evidence for a pathogenic splicing variant contributing to a rare disease and to identify tumor-specific splice isoforms associated with an oncogenic mutation. Bisbee was able to rediscover previously validated results in both of these cases and also identify common tumor-associated splice isoforms replicated in two independent melanoma datasets.

Aminoacyl tRNA synthetases (ARSs) link specific amino acids with their cognate transfer RNAs in a critical early step of protein translation. Mutations in ARSs have emerged as a cause of recessive, often complex neurological disease traits. Here we report an allelic series consisting of seven novel and two previously reported biallelic variants in valyl-tRNA synthetase (VARS) in ten patients with a developmental encephalopathy with microcephaly, often associated with early-onset epilepsy. In silico, in vitro, and yeast complementation assays demonstrate that the underlying pathomechanism of these mutations is most likely a loss of protein function. Zebrafish modeling accurately recapitulated some of the key neurological disease traits. These results provide both genetic and biological insights into neurodevelopmental disease and pave the way for further in-depth research on ARS related recessive disorders and precision therapies.

Inflammation in the diabetic retina is mediated by leukocyte adhesion to the retinal vasculature and alteration of the blood-retinal barrier (BRB). We investigated the role of chemokines in the alteration of the BRB in diabetes. Animals were made diabetic by streptozotocin injection and analyzed for gene expression and monocyte/macrophage infiltration. The expression of CCL2 (chemokine ligand 2) was significantly up-regulated in the retinas of rats with 4 and 8 weeks of diabetes and also in human retinal endothelial cells treated with high glucose and glucose flux. Additionally, diabetes or intraocular injection of recombinant CCL2 resulted in increased expression of the macrophage marker, F4/80. Cell culture impedance sensing studies showed that purified CCL2 was unable to alter the integrity of the human retinal endothelial cell barrier, whereas monocyte conditioned medium resulted in significant reduction in cell resistance, suggesting the relevance of CCL2 in early immune cell recruitment for subsequent barrier alterations. Further, using Cx3cr1-GFP mice, we found that intraocular injection of CCL2 increased retinal GFP+ monocyte/macrophage infiltration. When these mice were made diabetic, increased infiltration of monocytes/macrophages was also present in retinal tissues. Diabetes and CCL2 injection also induced activation of retinal microglia in these animals. Quantification by flow cytometry demonstrated a two-fold increase of CX3CR1+/CD11b+ (monocyte/macrophage and microglia) cells in retinas of wildtype diabetic animals in comparison to control non-diabetic ones. Using CCL2 knockout (Ccl2-/-) mice, we show a significant reduction in retinal vascular leakage and monocyte infiltration following induction of diabetes indicating the importance of this chemokine in alteration of the BRB. Thus, CCL2 may be an important therapeutic target for the treatment of diabetic macular edema.

Despite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf-Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood.