Aims: Following the seminal description of a de novo GABRA4 variant associated with early onset epilepsy and developmental delay, additional variants from patients and healthy controls are investigated.Methods: We collected molecular and phenotype data of individuals carrying de novo variants in GABRA4. A total of four patients and their parents were investigated either by exome or genome sequencing, followed by targeted Sanger sequencing in some cases. The variants are investigated utilizing molecular dynamics (MD) simulation studies, and functional studies using two electrode voltage clamp studies of recombinantly expressed, alpha4- containing GABAA receptors are performed.Results: Previously, we reported the de novo variant p.(Thr300Ile) in GABRA4 in a patient with epilepsy and neurodevelopmental abnormalities. The new patients with three novel de novo missense variants in GABRA4 display overlapping phenotypes among affected individuals including developmental delay (4/4), epileptiform EEG abnormalities (3/4), attention deficits (3/4), seizures (2/4), autistic features (2/4) and structural brain abnormalities (2/4). MD simulations of the three variants within the transmembrane domain of the receptor indicate that sub-microsecond scale dynamics differ between wild-type and mutated subunits. The functional studies require optimization due to the poor expression of the human alpha4 subunit in the X.l. oocyte expression system.Conclusions: Nine out of 19 genes encoding GABAA receptor subunits have been linked to monogenic syndromes characterized by seizures and developmental disorders previously. Taken together, our findings corroborate an association also between GABRA4 and a neurological phenotype including variable neurodevelopmental, behavioral and epileptic abnormalities.