Ion Channel–Rho GTPase Coupling in Human Odontogenesis: Integrative Structural and Network Dissection of TRIO and CACNA2D2 Variants From Familial Tooth Agenesis

INTRODUCTION: Tooth agenesis is a genetically heterogeneous developmental anomaly in which disturbances of morphogen signaling, cytoskeletal organization, and mineralization converge during odontogenesis. Building on a previously published clinical and exome study of two Lebanese families with familial nonsyndromic tooth agenesis that identified rare segregating missense variants in trio Rho guanine nucleotide exchange factor (TRIO) and calcium voltage-gated channel auxiliary subunit alpha2delta 2 (CACNA2D2), this work examines how these genes may participate in a shared mechanistic axis during human tooth development. METHODS: We performed a secondary, exclusively in silico mechanistic analysis of the originally reported variants, TRIO c.8312C>T (p.Ser2771Leu) and CACNA2D2 c.284G>A (p.Arg95His), without enrolling new participants or generating additional sequencing or clinical data. Protein domain organization was annotated from curated resources, residue-level and local-window conservation were quantified across vertebrate orthologs, and the structural context of each substituted residue was inspected using available experimental templates and full-length AlphaFold models from the AlphaFold Protein Structure Database. Population allele frequencies and clinical annotations were re-evaluated in large reference datasets, and high-confidence protein-protein interaction networks centered on TRIO, ras homolog family member A (RHOA), guanine nucleotide binding protein subunit beta 1 (GNB1), and CACNA2D2 were constructed to assess whether these genes form a common odontogenesis-relevant signaling landscape. RESULTS: Both substitutions localize to structured and functionally annotated regions: CACNA2D2 Arg95 lies in the extracellular N-terminal segment immediately upstream of the von Willebrand factor type A (VWA) domain, and TRIO Ser2771 lies within an immunoglobulin I-set domain adjacent to a C-terminal kinase-like region. In both cases, the affected residue is highly conserved across mammalian orthologs and falls within a locally conserved window. TRIO p.Ser2771Leu remains absent from population reference datasets, whereas CACNA2D2 p.Arg95His (reference SNP identifier, rs149979955) is a very rare allele with a non-dental Clinical Variants (ClinVar) annotation; both variants receive predominantly deleterious predictions. Structural inspection suggests possible effects on local packing, electrostatics, or interaction interfaces. Network analysis places TRIO, RHOA, GNB1, and CACNA2D2 along a high-confidence path enriched for cytoskeletal regulation, vesicle trafficking, calcium signaling, and mineralization, consistent with an ion channel-Rho guanosine triphosphatase (GTPase) coupling model in odontogenesis. CONCLUSIONS: This structural and network analysis suggests that the reported TRIO and CACNA2D2 variants may contribute to a testable TRIO-RHOA-GNB1-CACNA2D2 axis in human tooth development. The evidence remains hypothesis-generating and does not establish pathogenicity or causality, but it provides a coherent framework for targeted functional studies in dental and craniofacial models.