ABSTRACT The interactions between transcription factor (TFs) and their DNA binding sites are an integral part of the gene regulatory networks within cells. These interactions control critical steps in development and cell cycle control. Mutations in TFs can result in a wide range of structural birth defects or cancers through the dysregulation of gene expression. The Pediatric Data Resource Center established by the Gabriella Miller Kids First (GMKF) Pediatric Research Program (Kids First) provides genome sequence and phenotype data for studies investigating the genetics of childhood cancers or structural birth defects. Analysis of the GMKF data are needed to identify and annotate genetic variants associated with these major pediatric diseases. The goals of this project are: (1) to analyze genome sequence data from patients with structural birth defects made available as part of the Kids First Data Resource to identify genetic variants predicted to damage the ability of TFs to bind their DNA target sequences; and (2) to perform biochemical assays on a prioritized set of the TF coding variants to characterize their putatively damaged DNA binding activities. We anticipate our results will help to identify pathogenic variants contributing to structural birth defects or pediatric cancers, reveal mechanisms by which such variants may dysregulate gene expression leading to these disorders, lead to refined genomic diagnostics, and provide data on the mutant TFs’ altered DNA binding activities as a resource to the community.