Bi-allelic GAD2 variants cause a rare developmental encephalopathy with early-onset seizures

GAD67 and GAD65, encoded by GAD1 and GAD2, respectively, are the two isoforms of glutamic acid decarboxylase (GAD), the enzyme responsible for γ-aminobutyric acid (GABA) synthesis. Pathogenic variants in GAD1 cause a severe developmental and epileptic encephalopathy, whereas GAD2 has not previously been implicated in human disease.

We identified four individuals from three unrelated families with bi-allelic GAD2 loss-of-function variants. Clinical and genetic findings were combined with functional analysis of the variants. To further establish causality, we characterized a GAD65 knockout (GAD65-/-) mouse model for seizures, behavior and transcriptomic changes.

All individuals presented with early-onset epilepsy, developmental delay, mild to moderate intellectual disability and autism spectrum disorder. Seizures were most severe during infancy and appeared to diminish with age. EEG showed preserved background activity without consistent epileptiform abnormalities, and brain MRI revealed only minimal structural changes. All variants resulted in truncated protein products, supporting a recessive loss-of-function mechanism. GAD65-/- mice were viable and developed spontaneous seizures, with onset as early as four weeks and showed increased susceptibility to pentylenetetrazole (PTZ) induced seizures. These mice also exhibited increased locomotor, anxiety-like and repetitive behaviors, as well as social deficits and impairments in spatial learning and memory. Transcriptomic profiling revealed dysregulation of metabolic and mitochondrial pathways implicated in epilepsy.

Our findings firmly establish GAD2 as a disease gene implicated in developmental encephalopathy with early-onset seizures. The concordance between the human phenotype and the characteristics of the knockout mouse model emphasizes the pivotal role of GAD2 in neurodevelopment and epilepsy.