Mutations in have frequently been reported as etiologic for tooth agenesis (TA). However, the effects of variation on gene/protein function and contribution to TA phenotypes remain poorly understood. Here, we performed bioinformatic and functional characterization analysis of variants. In silico prediction of variant function was performed with VIPUR for all missense variants reported in the Exome Aggregation Consortium database. Functional characterization experiments were then performed for selected variants previously associated with TA. Expression vectors for wild-type and mutant were made and transfected into stem cells from human exfoliated deciduous teeth (SHED) for evaluation of gene/protein function, WNT signaling activity, and effects on expression of relevant genes. While 75% of variants were predicted neutral, most of the TA-associated variants received deleterious scores by potentially destabilizing or preventing the disulfide bond formation required for proper protein function. WNT signaling was significantly decreased with 8 of 13 variants tested, whereas wild-type-like activity was retained with 4 of 13 variants. mutant cells (T357I, R360C, and R379C mutants) showed reduced or impaired binding affinity to FZD5, suggesting a potential mechanism for the decreased WNT signaling. Mutant cells also had decreased WNT10A protein expression in comparison to wild-type cells. mRNA expression of , and (known tooth development genes) was perturbed in mutant cells and quite significantly for and . Transcriptome analysis of wild-type and T357I-mutant cells identified 36 differentially expressed genes (26 downregulated, 10 upregulated) involved in skeletal system development and morphogenesis and pattern specification. variants deemed pathogenic for TA likely affect protein folding and/or stabilization, leading to decreased WNT signaling and concomitant dysregulated expression of relevant genes. These findings may allow for improved interpretation of TA phenotypes upon clinical diagnosis while providing important insights toward the development of future tooth replacement therapies.