Defective neuronal migration during embryogenesis results in the cortical deformity known as lissencephaly (LIS), characterized by a flat or virtually smooth cerebral surface and a lack of gyral and sulcal development. Among the many gene products involved in neuronal migration is the 1 connected with the PAFAH1B1 gene. LIS was an uncommon disease with non-specific clinical signs, including early-onset epilepsy, developmental delay, or cerebral palsy-like movement difficulties, and a low prevalence in the general population. As high-throughput sequencing has been shown to improve diagnostic accuracy, the researchers decided to use this method to treat this patient. Herein, they reported the case of a 7-year-old male with non-specific clinical signs probably compatible with lissencephaly, a rare condition for which no diagnosis was made. The patient participated in a study that required whole-genome sequencing. A bioinformatics workflow was used to decipher the sequence data. After collecting variants, they were annotated and prioritized using a series of criteria-based filters. Mitochondrial DNA was examined as well. The index case phenotype was explained by identifying a unique potential frameshift insertion in the well-studied PAFAH1B1 gene. As determined by in silico analysis, it was harmful and led to nonsense-mediated processes, earning a high degree of certainty in the assessment. The insertion shifts the reading frame, leading to a premature stop codon and, most likely, the silencing of one allele, which both have devastating effects on the protein’s function. Neither the mother nor the father was carriers of the mutation, and the latter was not accessible for testing. This investigation identified a unique de novo mutation in the LIS1/PAFAH1B1 gene as the likely cause of a rare condition in a young child with non-specific clinical signs. Since the loss of function in the gene product has been previously characterized in this condition, the mutation discovered coincides with the phenotype examined. The absence of other rare mutations in PAFAH1B1 and the presence of a strong family history support the hypothesis of a de novo illness mechanism.