Genetically tractable fruit flies have been used for decades to study seizure disorders. However, there is a paucity of data specifically correlating fly and human seizure phenotypes. We have previously shown that mutation of orthologous PRICKLE genes from flies to humans produce seizures. This study aimed to determine whether the prickle-mediated seizure phenotypes in flies closely parallel the epilepsy syndrome found in PRICKLE patients.

Receptor occupancy (RO) assays provide a means to measure the direct interaction of therapeutics with their cell surface targets. Free receptor assays quantify cell-surface receptors not bound by a therapeutic while total receptor assays quantify the amount of target on the cell surface.

Animal models of COVID-19 have been rapidly reported after the start of the pandemic. We aimed to assess whether the newly created models reproduce the full spectrum of human COVID-19.

Maffucci syndrome is a rare, highly variable somatic mosaic condition, and well-known cancer-related gain-of-function variants in either the IDH1 or IDH2 genes have been found in the affected tissues of most reported individuals. Features include benign enchondroma and spindle-cell hemangioma, with a recognized increased risk of various malignancies. Fewer than 200 affected individuals have been reported; therefore, accurate estimates of malignancy risk are difficult to quantify and recommended surveillance guidelines are not available. The same gain-of-function IDH1 and IDH2 variants are also implicated in a variety of other benign and malignant tumors. An adult male presented with several soft palpable lesions on the right upper limb. Imaging and histopathology raised the possibility of Maffucci syndrome. DNA was extracted from peripheral blood lymphocytes and tissue surgically resected from a spindle-cell hemangioma. Sanger sequencing and droplet digital polymerase chain reaction (PCR) analysis of the IDH1 gene were performed. We identified a somatic mosaic c.394C > T (p.R132C) variant in exon 5 of IDH1, in DNA derived from hemangioma tissue at ∼17% variant allele fraction. This variant was absent in DNA derived from blood. This variant has been identified in the affected tissue of most reported individuals with Maffucci syndrome. Although this individual has a potentially targetable variant, and there is a recognized risk of malignant transformation in this condition, a decision was made not to intervene with an IDH1 inhibitor. The reasons and prospects for therapy in this condition are discussed.

Biopharmaceuticals administered to the human body have the potential to trigger the production of anti-drug (also called anti-therapeutic) antibodies (ADA) that can neutralize the therapeutic activity. For antibody therapeutics, cell-based neutralizing ADA assays are frequently used to evaluate ADA in clinical studies. We developed a method to detect neutralizing antibodies against MEDI-575, a fully human IgG2κ antagonistic antibody against PDGFR-α. We evaluated three assay formats, two of which measured late responses, cell proliferation and apoptosis, whereas the third assay detected an early signaling event, phosphorylation of PDGFR-α. Measuring phosphorylation provided a superior assay window and therefore was developed as a neutralizing ADA (NAb) assay. Matrix interference, however, was significant, and could be identified to be caused by PDGF-AA and PDGF-AB, apparently the two most abundant ligands of PDGFR-α present in human serum samples. A simple pre-treatment step, addition of an inhibitory antibody to PDGF-A, a subunit present in PDGF-AA and PDGF-AB, was found to eliminate matrix interference, increasing assay reliability and sensitivity. We integrated the pre-treatment step into assay development and qualified a robust NAb assay.

Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy.

Interferon-induced membrane proteins (IFITM) 3 gene variants are known risk factor for severe viral diseases. We examined whether IFITM3 variant may underlie the heterogeneous clinical outcomes of SARS-CoV-2 infection-induced COVID-19 in large Arab population. We genotyped 880 Saudi patients; 93.8% were PCR-confirmed SARS-CoV-2 infection, encompassing most COVID-19 phenotypes. Mortality at 90 days was 9.1%. IFITM3-SNP, rs12252-G allele was associated with hospital admission (OR = 1.65 [95% CI; 1.01-2.70], P = 0.04]) and mortality (OR = 2.2 [95% CI; 1.16-4.20], P = 0.01). Patients less than 60 years old had a lower survival probability if they harbor this allele (log-rank test P = 0.002). Plasma levels of IFNγ were significantly lower in a subset of patients with AG/GG genotypes than patients with AA genotype (P = 0.00016). Early identification of these individuals at higher risk of death may inform precision public health response.

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 prickle mutant flies by the small-molecule USP9X inhibitor, Degrasyn/WP1130, or by reducing the dose of fat facets a USP9X orthologue. USP9X mutations were identified by resequencing a cohort of patients with epileptic encephalopathy, one patient harbored a de novo missense mutation and another a novel coding mutation. Both USP9X variants were outside the PRICKLE-interacting domain. These findings demonstrate that USP9X inhibition can suppress prickle-mediated seizure activity, and that USP9X 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.

GAT-1, encoded by SLC6A1, is one of the major gamma-aminobutyric acid (GABA) transporters in the brain and is responsible for re-uptake of GABA from the synapse. In this study, targeted resequencing of 644 individuals with epileptic encephalopathies led to the identification of six SLC6A1 mutations in seven individuals, all of whom have epilepsy with myoclonic-atonic seizures (MAE). We describe two truncations and four missense alterations, all of which most likely lead to loss of function of GAT-1 and thus reduced GABA re-uptake from the synapse. These individuals share many of the electrophysiological properties of Gat1-deficient mice, including spontaneous spike-wave discharges. Overall, pathogenic mutations occurred in 6/160 individuals with MAE, accounting for ~4% of unsolved MAE cases.

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution.

The WWOX gene has a WW domain containing oxidoreductase, which is located at the common fragile site FRA16D at chromosome 16q23. WWOX is a tumor suppressor gene that has been associated with several types of cancer such as hepatic, breast, lung, prostate, gastric, and ovarian. Recently WWOX has been implicated in epilepsy, where studies show homozygous loss-of-function mutation lead to early-infantile epileptic encephalopathy, spinocerebellar ataxia, intractable seizures and developmental delay, and early lethal microcephaly syndrome with epilepsy. Here we investigate two consanguineous Saudi families and we identified three probands with epileptic encephalopathy. Whole exome sequencing revealed a novel homozygous mutation in the WWOX gene in one proband. In addition, we identified a previously reported WWOX mutation in two probands. Later on these findings were confirmed with Sanger sequencing. The underlying mechanism on how WWOX mutations lead to seizure remains elusive. To date very few WWOX mutations have been associated with neurological disorder and our newly identified mutations support the notion that WWOX play an important role in neurons and will aid in better diagnosis and genetic counseling.

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, PRICKLE1 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 Prickle1(+/-) mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 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 SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.

Brain somatic mutations are an increasingly recognized cause of epilepsy, brain malformations and autism spectrum disorders and may be a hidden cause of other neurodevelopmental and neurodegenerative disorders. At present, brain mosaicism can be detected only in the rare situations of autopsy or brain biopsy. Liquid biopsy using cell-free DNA derived from cerebrospinal fluid has detected somatic mutations in malignant brain tumours. Here, we asked if cerebrospinal fluid liquid biopsy can be used to detect somatic mosaicism in non-malignant brain diseases. First, we reliably quantified cerebrospinal fluid cell-free DNA in 28 patients with focal epilepsy and 28 controls using droplet digital PCR. Then, in three patients we identified somatic mutations in cerebrospinal fluid: in one patient with subcortical band heterotopia the LIS1 p. Lys64* variant at 9.4% frequency; in a second patient with focal cortical dysplasia the TSC1 p. Phe581His*6 variant at 7.8% frequency; and in a third patient with ganglioglioma the BRAF p. Val600Glu variant at 3.2% frequency. To determine if cerebrospinal fluid cell-free DNA was brain-derived, whole-genome bisulphite sequencing was performed and brain-specific DNA methylation patterns were found to be significantly enriched (P = 0.03). Our proof of principle study shows that cerebrospinal fluid liquid biopsy is valuable in investigating mosaic neurological disorders where brain tissue is unavailable.