We describe two sisters from a consanguineous Arab family with global developmental delay, dystrophy, axial hypotonia, epileptic encephalopathy dominated by intractable complex partial seizures that were resistant to various anti-epileptic treatments. Dysmorphic features comprised low set ears, hypertelorism, upslanting palpebral fissures, a broad nasal bridge, and blue sclera with elongated eyelashes. Brain MRI in both children showed a corpus callosum hypoplasia that was evident already in utero and evolving cortical atrophy. Autozygosity mapping in combination with Whole Exome Sequencing revealed a homozygous missense mutation in the PIGO gene [c.765G > A, NM_032634.3] that affected a highly conserved methionine in the alkaline phosphatase-like core domain of the protein [p.(Met255Ile), NP_116023.2]. PIGO encodes the GPI-ethanolamine phosphate transferase 3, which is crucial for the final synthetic step of the glycosylphosphatidylinositol-anchor that attaches many enzymes to their cell surfaces, such as the alkaline phosphatase and granulocyte surface markers. Interestingly, measurement of serum alkaline phosphatase activities in both children was normal or only slightly elevated. Quantification of granulocyte surface antigens CD16/24/59 yielded reduced levels only for CD59. Phenotype analysis of our and other published patients with PIGO mutations reveals a more severe affectation and predominantly neurological presentation in individuals carrying a mutation in the alkaline phosphatase-like core domain thereby hinting towards a genotype-phenotype relation for PIGO gene mutations.