Novel Ectodysplasin-A Variants: Structural and Functional Basis of Hypohidrotic Ectodermal Dysplasia

Abstract This study investigates two novel variants in the EDA, c.680G > A (p.G227E) and c.649_666del (Δ215–220), identified in X-linked ectodermal dysplasia and syndromic tooth agenesis cases. These variants were identified through Sanger sequencing and mapped to highly conserved regions of EDA. Bioinformatics tools consistently classified them as deleterious, with significant disruptions predicted in protein stability, hydrophobicity, and secondary structure. Structural analysis revealed that p.G227E caused a glycine-to-glutamic acid substitution, altering hydrophobicity and secondary structure, while Δ215–220 disrupted a conserved hydrophobic region, leading to increased protein instability Functional studies revealed reduced expression of EDA and WNT4 proteins, alongside increased IκB levels and decreased NF-κB mRNA expression, indicating impaired EDA-NF-κB signaling. Subcellular localization analyses demonstrated diminished cytoplasmic expression of the EDA Variants proteins, corroborated by in silico predictions. Post-translational modifications (PTMs) and gene ontology (GO) analyses revealed alterations in processes critical for ectodermal development, including macromolecule biosynthesis, nitrogen metabolism, and receptor signaling. Molecular dynamics simulations highlighted increased rigidity, compact structure, and reduced flexibility in the EDA variants proteins compared to EDA Wild Type (WT). Interestingly, neither variant significantly impacted calcium or mitochondrial potential under normal experimental conditions, suggesting their pathogenic effects arise primarily from disrupted protein interactions and signaling pathways. This study integrates molecular, bioinformatics, and functional analyses to elucidate the pathogenicity of these novel EDA variants, providing insights into ectodermal dysplasia mechanisms and paving the way for future therapeutic strategies targeting these EDA variants.