Introduction: The NPRL3 gene is a critical component of the GATOR1 complex, which negatively regulates the mTORC1 pathway, essential for neurogenesis and brain development. Located on chromosome 16p13.3, NPRL3 is situated near the α-globin gene cluster. Haploinsufficiency of NPRL3, either by deletion or a pathogenic variant, is associated with a variable phenotype of focal epilepsy, with or without malformations of cortical development, with known decreased penetrance. Case Description: This work details the diagnostic odyssey of a neurotypical 10-year-old boy who presented at age 2 with unusual nocturnal episodes and a history of microcytic anemia, as well as a review of the existing literature on NPRL3-related epilepsy, with an emphasis on individuals with deletions who also present with α-thalassemia trait. The proband\'s episodes were mistaken for gastroesophageal reflux disease for several years. He had molecular testing for his α-thalassemia trait and was noted to carry a deletion encompassing the regulatory region of the α-thalassemia gene cluster. Following the onset of overt focal motor seizures, genetic testing revealed a heterozygous loss of NPRL3, within a 106 kb microdeletion on chromosome 16p13.3, inherited from his mother. This deletion encompassed the entire NPRL3 gene, which overlaps the regulatory region of the α-globin gene cluster, giving him the dual diagnosis of NPRL3-related epilepsy and α-thalassemia trait. Brain imaging postprocessing showed left hippocampal sclerosis and mid-posterior para-hippocampal focal cortical dysplasia, leading to the consideration of epilepsy surgery. Conclusions: This case underscores the necessity of early and comprehensive genetic assessments in children with epilepsy accompanied by systemic features, even in the absence of a family history of epilepsy or a developmental delay. Recognizing phenotypic overlaps is crucial to avoid diagnostic delays. Our findings also highlight the impact of disruptions in regulatory regions in genetic disorders: any individual with full gene deletion of NPRL3 would have, at a minimum, α-thalassemia trait, due to the presence of the major regulatory element of α-globin genes overlapping the gene\'s introns.

GATOR1 (GAP Activity TOward Rag 1) is an evolutionarily conserved GTPase-activating protein complex that controls the activity of mTORC1 (mammalian Target Of Rapamycin Complex 1) in response to amino acid availability in cells. Genetic mutations in the GATOR1 subunits, NPRL2 (nitrogen permease regulator-like 2), NPRL3 (nitrogen permease regulator-like 3), and DEPDC5 (DEP domain containing 5), have been associated with epilepsy in humans; however, the specific effects of these mutations on GATOR1 function and mTORC1 regulation are not well understood. Herein, we report that epilepsy-linked mutations in the NPRL2 subunit of GATOR1, NPRL2-L105P, -T110S, and -D214H, increase basal mTORC1 signal transduction in cells. Notably, we show that NPRL2-L105P is a loss-of-function mutation that disrupts protein interactions with NPRL3 and DEPDC5, impairing GATOR1 complex assembly and resulting in high mTORC1 activity even under conditions of amino acid deprivation. Furthermore, our studies reveal that the GATOR1 complex is necessary for the rapid and robust inhibition of mTORC1 in response to growth factor withdrawal or pharmacological inhibition of phosphatidylinositol-3 kinase (PI3K). In the absence of the GATOR1 complex, cells are refractory to PI3K-dependent inhibition of mTORC1, permitting sustained translation and restricting the nuclear localization of TFEB, a transcription factor regulated by mTORC1. Collectively, our results show that epilepsy-linked mutations in NPRL2 can block GATOR1 complex assembly and restrict the appropriate regulation of mTORC1 by canonical PI3K-dependent growth factor signaling in the presence or absence of amino acids.

We evaluated the utility of genetic testing in the pre-surgical evaluation of pediatric patients with drug-resistant focal epilepsy. This single-center retrospective study reviewed the charts of all pediatric patients referred for epilepsy surgery evaluation over a 5-year period. We extracted and analyzed results of genetic testing as well as clinical, EEG, and neuroimaging data. Of 125 patients referred for epilepsy surgical evaluation, 86 (69%) had some form of genetic testing. Of these, 18 (21%) had a pathogenic or likely pathogenic variant identified. Genes affected included NPRL3 (3 patients, all related), TSC2 (3 patients), KCNH1, CHRNA4, SPTAN1, DEPDC5, SCN2A, ARX, SCN1A, DLG4, and ST5. One patient had ring chromosome 20, one a 7.17p12 duplication, and one a 15q13 deletion. In six patients, suspected epileptogenic lesions were identified on brain MRI that were thought to be unrelated to the genetic finding. A specific medical therapy choice was allowed due to genetic diagnosis in three patients who did not undergo surgery. Obtaining a molecular diagnosis may dramatically alter management in pediatric patients with drug-resistant focal epilepsy. Genetic testing should be incorporated as part of standard investigations in the pre-surgical work-up of pediatric patients with drug-resistant focal epilepsy.

Focal epilepsy accounts for 60% of all forms of epilepsy, but the pathogenic mechanism is not well understood. In this study, three novel mutations in NPRL3 (nitrogen permease regulator-like 3), c.937_945del, c.1514dupC and 6,706-bp genomic DNA (gDNA) deletion, were identified in three families with focal epilepsy by linkage analysis, whole exome sequencing (WES) and Sanger sequencing. NPRL3 protein is a component of the GATOR1 complex, a major inhibitor of mTOR signaling. These mutations led to truncation of the NPRL3 protein and hampered the binding between NPRL3 and DEPDC5, which is another component of the GATOR1 complex. Consequently, the mutant proteins enhanced mTOR signaling in cultured cells, possibly due to impaired inhibition of mTORC1 by GATOR1. Knockdown of nprl3 in Drosophila resulted in epilepsy-like behavior and abnormal synaptic development. Taken together, these findings expand the genotypic spectrum of NPRL3-associated focal epilepsy and provide further insight into how NPRL3 mutations lead to epilepsy.

UNASSIGNED: As one of the assembly factors of the GATOR1 protein complex in the mechanism of rapamycin pathway, NPRL3 plays an important role in the pathogenesis of epilepsy. However, the correlation between genotype and clinical phenotype in patients with NPRL3-related epilepsy has not been clarified.
UNASSIGNED: A total of 11 Chinese children with NPRL3-related epilepsy were identified through whole-exome sequencing (WES). The data from the clinical presentation, laboratory data, brain imaging findings, genetic results, and treatment methods were collected. All previously reported cases with NPRL3-related epilepsy were collected and reviewed through PubMed search.
UNASSIGNED: Among the 11 children, eight have not been reported, and two of them presented infantile spasms (ISs) as a new phenotype of NPRL3-related epilepsy. In addition, WES identified five frameshift mutations, three nonsense mutations, two missense mutations, and one exon deletion. Based on bioinformatics analysis, it was found that two missense mutation sites were highly conserved, and the c.400G>A mutation site of the NPRL3 gene caused the alteration of the protein structure. To date, 88 patients have been reported with NPRL3-related defects, including our 11 cases. The most common presentations were sleep-related hypermotor epilepsy (SHE), frontal lobe epilepsy (FLE), and temporal lobe epilepsy. A majority of patients (70%) presented normal neuroimaging results, and focal cortical dysplasia was the most common neuroimaging abnormality (62.5%). Among the NPRL3 gene mutations, loss of function (nonsense mutations, frameshift mutations, and exons deletion) was the most common genetic variation (75%). For 73% of patients with NPRL3-related epilepsy, monotherapy of sodium channel blockers was effective. Surgery was effective for 75% of children with neuroimaging abnormalities. Two cases unresponsive to surgery or anti-seizure medications were treated with ketogenic diets (KD), which were effective. One case was treated with rapamycin at an early stage of epilepsy, which was effective as well.
UNASSIGNED: NPRL3-related epilepsy has high clinical and genetic heterogeneity. SHE and FLE are the most common clinical presentations. Furthermore, ISs are the new phenotypes of NPRL3-related epilepsy, while the variants c.275G>A, c.745G>A, and c.1270C>T may be the most common NPRL3 gene mutations. Sodium channel blockers, surgery, KD, and rapamycin may be the potential treatments for these patients. Our study expanded the clinical and genetic spectrum of NPRL3-related epilepsy and provided important information for the precise treatment of patients.

Variants in GATOR1 genes are well established in focal epilepsy syndromes. A strong association of GATOR1 variants with drug-resistant epilepsy as well as an increased risk of sudden unexplained death in epilepsy warrants developing strategies to facilitate the identification of patients who could potentially benefit from genetic testing and precision medicine. We aimed to determine the yield of GATOR1 gene sequencing in patients with focal epilepsy typically referred for genetic testing, establish novel GATOR1 variants and determine clinical, electroencephalographic, and radiological characteristics of variant carriers.
Ninety-six patients with clinical suspicion of genetic focal epilepsy with previous comprehensive diagnostic epilepsy evaluation in The Neurology Clinic, University Clinical Center of Serbia, were included in the study. Sequencing was performed using a custom gene panel encompassing DEPDC5, NPRL2, and NPRL3. Variants of interest (VOI) were classified according to criteria proposed by the American College of Medical Genetics and the Association for Molecular Pathology.
Four previously unreported VOI in 4/96 (4.2%) patients were found in our cohort. Three likely pathogenic variants were determined in 3/96 (3.1%) patients, one frameshift variant in DEPDC5 in a patient with nonlesional frontal lobe epilepsy, one splicogenic DEPDC5 variant in a patient with nonlesional posterior quadrant epilepsy, and one frameshift variant in NPRL2 in a patient with temporal lobe epilepsy associated with hippocampal sclerosis. Only one VOI, a missense variant in NPRL3, found in 1/96 (1.1%) patients, was classified as a variant of unknown significance.
GATOR1 gene sequencing was diagnostic in 3.1% of our cohort and revealed three novel likely pathogenic variants, including a previously unreported association of temporal lobe epilepsy with hippocampal sclerosis with an NPRL2 variant. Further research is essential for a better understanding of the clinical scope of GATOR1 gene-associated epilepsy.

Background: The GAP Activity Towards Rags 1 (GATOR1) complex, which includes DEPDC5, NPRL2, and NPRL3, plays a key role in epilepsy. It has been reported that focal epilepsy is associated with mutations in the NPRL3 gene in some cases. We report two rare mutations in the NPRL3 gene in two unrelated Chinese families with focal epilepsy in this study. Methods: The proband and her brother in family E1 first experienced seizures at 1.5 and 6 years of age, respectively. Despite resection of epileptogenic foci, she still suffered recurrent seizures. The first seizure of a 20-year-old male proband in family E2 occurred when he was 2 years old. To identify pathogenic variants in these families, whole-exome sequencing (WES) was performed on genomic DNA from peripheral blood. Results: In family E1, the trio-WES analysis of the proband and her brother without apparent structural brain abnormalities identified a heterozygous variant in the NPRL3 gene (c.954C>A, p.Y318*, NM_001077350.3). In family E2, the proband carried a heterozygous NPRL3 mutation (c.1545-1G>C, NM_001077350.3). Surprisingly, the mothers of the two probands each carried the variants, but neither had an attack. Bioinformatics analysis predicted that the mutation (c.954C>A) was in the highly conserved amino acid residues of NPRL3, which affected the α-helix of NPRL3 protein, leading to a truncated protein. The splice variant (c.1545-1G>C) resulted in the loss of the last exon of the NPRL3 gene. Conclusion: The results of this study provide a foundation for diagnosing NPRL3-related epilepsy by enriching their genotypes and phenotypes and help us identify the genetic etiologies of epilepsy in these two families.

Mutations in the GATOR1 complex genes, DEPDC5 and NPRL3, play a major role in the development of lesional and non-lesional focal epilepsy through increased mTORC1 signalling. We aimed to assess the effects of mTORC1 hyperactivation on GABAergic inhibitory circuits, in 3 and 5 individuals carrying DEPDC5 and NPRL3 mutations respectively using a multimodal approach including transcranial magnetic stimulation (TMS), magnetic resonance spectroscopy (MRS), and electroencephalography (EEG).
Inhibitory functions probed by TMS and MRS showed no effect of mutations on cortical GABAergic receptor-mediated inhibition and GABA concentration, in both cortical and subcortical regions. However, stronger EEG theta oscillations and stronger and more synchronous gamma oscillations were observed in DEPDC5 and NPRL3 mutations carriers.
These results suggest that DEPDC5 and NPRL3-related epileptic mTORopathies may not directly modulate GABAergic functions but are nonetheless characterized by a stronger neural entrainment that may be reflective of a cortical hyperexcitability mediated by increased mTORC1 signaling.

Familial focal epilepsy with variable foci is an autosomal dominant disorder characterized by partial epilepsy with variable foci. In this study, we report a six-generation with segregation of the mutation present in four generations Chinese family presenting with focal epilepsy with variable foci. Whole exome sequencing confirms a novel pathogenic mutation in the NPRL3 gene (c316C>T; p. Q106*). PCR, Western blotting, and immunohistochemistry were conducted to analyze the gene transcription, protein expression, and subcellular localization of NPRL3 and related signaling molecules in peripheral blood cells from family members. As compared with healthy family members, both mRNA level and protein expression of NPRL3 are decreased in peripheral blood cells of the mutation carrier. In addition, the expression of downstream molecular Phospho-p70 S6 kinase (P-s6k) are increased consequently. Our findings expand the genotypic and phenotypic spectrum of the NPRL3-associated epilepsy and reveal the mechanisms of mTOR pathway signaling and GATOR1 pathogenesis in focal epilepsies, providing exciting potential for future diagnostic and therapeutic interventions. However, further in vitro and animal experiments are still needed to evaluate the role of NPRL3 loss-of-function mutation in epileptogensis.

Introduction: NPRL3 gene mutations cause autosomal dominant familial focal epilepsy of variable foci (FFEVF) and is characterized by focal epilepsy arising from different brain regions including temporal, frontal, parietal and occipital lobes. About 50% of patients with NPRL3 related epilepsy are resistant to medical treatment. Method: We present a case of 27 years old man with NPRL3 related focal drug-resistant epilepsy. Stereotactic EEG showed two independent seizure foci, namely, left hippocampus and left orbitofrontal cortices. He underwent laser interstitial thermal therapy for ablating both foci in the same procedure that led to seizure cessation. Conclusion: laser interstitial thermal therapy can be an effective treatment for drug resistant NPRL3 related focal epilepsy with better tolerance and less morbidity as compared to open surgical resection, particularly in those with multiple seizure foci.