Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder with a strong genetic component. Although next-generation sequencing (NGS) technologies have been successfully applied to gene identification in de novo ASD, the genetic architecture of familial ASD remains largely unexplored. Our approach, which leverages the high specificity and sensitivity of NGS technology, has focused on rare variants in familial autism. We used NGS exome sequencing in 26 families with distantly related affected individuals to identify genes with private gene disrupting and missense variants of interest (VOI). We found that the genes carrying VOIs were enriched for biological processes related to cell projection organization and neuron development, which is consistent with the neurodevelopmental hypothesis of ASD. For a subset of genes carrying VOIs, we then used targeted NGS sequencing and gene-based variant burden case-control analysis to test for association with ASD. Missense variants in one gene, CEP41, associated significantly with ASD (p = 6.185e-05). Homozygous gene-disrupting variants in CEP41 were initially found to be responsible for recessive Joubert syndrome. Using a zebrafish model, we evaluated the mechanism by which the CEP41 variants might contribute to ASD. We found that CEP41 missense variants affect development of the axonal tract, cranial neural crest migration and social behavior phenotype. Our work demonstrates the involvement of CEP41 heterozygous missense variants in ASD and that biological processes involved in cell projection organization and neuron development are enriched in ASD families we have studied.