Objective: Glucose transporter-1 deficiency syndrome (GLUT1-DS) is defined as a metabolic encephalopathy that is associatedwith heterozygous and usually de novo pathogenic variations inthe SLC2A1 (solute carrier family2 member1) gene.Materials and Methods: In this study, all coding exons andneighboring intronic regions of SLC2A1 were Sanger sequencedin 12 patients with clinically suspected GLUT1-DS. For de novovariations revealed after sequencing and segregation analysis,we also performed genome wide Single Nucleotide Polymorphism(SNP) genotyping to confirm parental relatedness withthe proband. In patients without any sequence variations, real-time quantitative real-time polymerase chain reaction (qPCR)was applied to determine the presence of any copy number variations(CNV).Results: Sanger sequencing followed by bioinformatics analysis,segregation in the family and SNP array genotyping revealedtwo novel and de novo pathogenic variations associated withthe GLUT1-DS phenotype in 2 patients. qPCR results were compatible with one copy loss of SLC2A1 gene in another patient.All variations identified herein are likely to have caused null allelesand resulted in GLUT1-DS through haplo insufficiency.Disscussion: In this study we used a series of molecular geneticapproaches in order to identify all possible variations in SLC2A1that may be associated with GLUT1-DS. This collective effort facilitateddiagnosis in 3 patients.