Everolimus precision therapy for the GATOR1‐related epilepsies: A case series

Pathogenic variants in the GAP activity towards RAGs 1 (GATOR1) complex genes (DEPDC5, NPRL2, NPRL3) cause focal epilepsy through hyperactivation of the mechanistic target of rapamycin pathway. We report our experience using everolimus in patients with refractory GATOR1‐related epilepsy.

identification of somatic DEPDC5 variants in resected FCD tissue from patients with germline DEPDC5 variants provides evidence for a 'two-hit' mechanism [3,4]. Several studies also report an increased risk of sudden unexpected death in epilepsy in GATOR1-related epilepsies [1,5].
Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that combines with several proteins to form mTOR complex 1 (mTORC1), a pivotal regulator of cell growth and metabolism. GATOR1 complex inhibits mTORC1 in response to intracellular amino acid levels [6]. Molecular evidence of mTORC1 hyperactivation has been observed in resected brain tissue from patients with loss-of-function (LoF) variants in DEPDC5 and NPRL3 [2,3].
Everolimus-a synthetic mTOR inhibitor-is an approved treatment for DRE in tuberous sclerosis complex (TSC), the prototypical mTORopathy. Analogous to TSC, mTORC1 hyperactivity appears to be a driver of seizures in GATOR1-related epilepsies. The demonstration of rapamycin-responsive seizures in 'GATORopathy' mouse models [7,8], coupled with the therapeutic success of everolimus for TSC-related epilepsy, has given rise to the hypothesis that mTOR inhibitors may ameliorate seizures in GATOR1-related epilepsy [9]. Here we report our experience using everolimus in refractory GATOR1-related epilepsy.

Patients with epilepsy caused by variants in DEPDC5, NPRL2
or NPRL3 were identified through genomic research at the FutureNeuro Research Centre and Children's Health Ireland at Temple Street, Dublin. Patients with pathogenic or likely pathogenic variants in GATOR1 genes as determined by American College of Medical Genomics (ACMG) criteria were included [9]. Patients with GATOR1 missense variants of uncertain significance (VUS) were considered for inclusion based on several factors including phenotype compatibility, frequency in population databases, in silico predictions and segregation analysis, following review at our epilepsy genetics multidisciplinary meeting (MDM). Confirmation genetic testing was conducted by an accredited service provider (CeGat GmbH), who also provided an independent interpretation of variant pathogenicity.
Nine patients with GATOR1-related epilepsy were identified, including six unrelated patients and three siblings. Five patients with GATOR1 variants had poorly controlled DRE, one experienced infrequent focal seizures, and three were seizure-free. The five patients with active epilepsy were offered a trial of everolimus. The remaining four patients, whose epilepsy was well controlled, were not considered for the everolimus trial. Demographic and epilepsyrelated data for patients not treated with everolimus are provided in  PrimateAI predicting a pathogenic impact [10,11]. No family history of seizures was reported. Segregation testing was limited to the patient's mother, who was negative for the DEPDC5 variant.
He had no siblings and his father was deceased. Following discussion at the epilepsy genetics MDM, we decided to treat Patient 5, given the compatibility of the epilepsy syndrome with the DEPDC5 phenotype, the rarity of the variant in gnomAD, and the severity of the patient's seizures and peri-ictal psychiatric symptoms.
The median duration of treatment was 12 months (IQR 19.5 months), with a median everolimus dose of 12.5 mg/day (IQR 5 mg/day). Patients 2, 3 and 5 had serum everolimus concentrations greater than 5 ng/mL (range 5.1-8.9 ng/mL) at last assessment. Patients 1 and 4 had everolimus levels less than 5 ng/mL but did not tolerate higher everolimus doses. Patients 1, 2 and 3 were treatment responders, all achieving a greater than 70% MMSF reduction from baseline (range 74.3%-86.1%) ( Table 2). The three responders had  Baseline seizure frequency was calculated as the mean number of seizures per month over the previous 3 months before starting everolimus.
c PVS1 refers to a "null variant (nonsense, frameshift, canonical ±1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss-of-function is a known mechanism of disease" [10].
PM2 is defined as absent from controls in the Exome Sequencing Project, the 1000 Genomes Project, or the Exome Aggregation Consortium [10]. PP5 is applied when a "reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation" [10]. PM1 is applied to a variant "located in a mutational hot spot and/or critical and wellestablished functional domain without benign variation" [10]. BP4 is applied to a variant when "multiple lines of computational evidence suggest no impact on gene or gene product" [10].

TA B L E 1 (Continued)
LoF variants and may induce seizures through mTORC1-independent mechanisms.
Potential reasons for treatment failure in Patient 4 included the subtarget everolimus level, the causal gene (NPRL3) and variant type (splicesite). The anti-seizure benefit of rapamycin was less impressive in Nprl3 knockout mice compared with Depdc5 knockout mice [8]. Sirolimus treatment produced varied outcomes in two infants with NPRL3-related hemimegalencephaly. One infant had reduced seizures but stopped treatment after 3.5 months due to recurrent infections [15], while the other stopped sirolimus after 17 days due to lack of efficacy [16]. in the same gene could facilitate larger precision therapy trials [9]. The 'n-of-1' multi-crossover trial design has been proposed as a more robust tool to investigate precision therapies in rare genetic disorders.
Notwithstanding these limitations, our findings suggest that everolimus is a promising precision treatment for GATOR1-related epilepsies, particularly DRE caused by DEPDC5 LoF variants.
Everolimus might be considered alongside high-efficacy treatments like epilepsy surgery for DEDPC5 mTORopathy, although larger studies are needed to support this recommendation.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.