Tooth agenesis (TA), failure to develop all permanent teeth, is the most prevalent dentofacial abnormality in humans (Al-Ani et al., 2017). The prevalence rate of TA is reported to range from 3% to 10% (Jonsson et al., 2018). TA conditions may appear as syndromic tooth agenesis (STA) or non-syndromic tooth agenesis (NSTA) according to whether accompanied by some systemic diseases (Chhabra et al., 2014).

Human tooth development is regulated by gene networks, in which signaling pathways involved in ectodermal organs play an important role. Disturbance of any balanced signaling cascade may lead to abnormal tooth development (Yu et al., 2019). The development of human tooth number and morphology is highly sensitive to changes in the ectodysplasin-A (EDA)/ectodysplasin A receptor (EDAR)/nuclear factor-κB (NF-κB) pathway (Han et al., 2018, Häärä et al., 2012). Three important components of the pathway are encoded by EDA (OMIM: 300451), EDAR (OMIM: 604095), ectodysplasin-A receptor-associated adapter (EDARADD, OMIM: 606603). EDAR, the type I transmembrane protein, belongs to the tumor necrosis factor receptor (TNFR) superfamily (Wang et al., 2020). Its primary structure includes the N-terminal ectodomain, transmembrane domain (TM), and C-terminal intracellular domain. The extracellular region has a cysteine-rich domain and a signal peptide, while the intracellular portion contains a death domain (DD) (Okita et al., 2019). EDAR has been demonstrated to interact with the secreted protein ectodysplasin-A1 (EDA-A1, one isoform of the ligand EDA) first (Mikkola & Thesleff, 2003), and subsequently, DD (residues from 358-431) in the intracellular cytoplasmic region of EDAR binds with DD (residues from 123-202) in EDARADD (Parveen et al., 2019, Sadier et al., 2014). The EDA/EDAR/EDARADD complex was reported to activate the NF-κB for the development of ectodermal organs, including teeth, hairs, feathers, and mammary glands (Cai et al., 2021, Yu et al., 2019). A majority of EDAR variants were found to result in hypohydrotic ectodermal dysplasia (HED)-related TA, however, only 21 EDAR variants have been identified in NSTA (Arte et al., 2013, Chen et al., 2017, Jonsson et al., 2018, Mumtaz et al., 2020, Yamaguchi et al., 2017, Zeng et al., 2017, Zhang et al., 2021, Zhang et al., 2020).

The conformational motion of protein can be tracked over time in atomic-level detail using molecular dynamics (MD) simulations (Collier et al., 2020). The molecular mechanics/generalized Born surface area (MM/GBSA) method is a common technique for estimating the free energy of the binding of biological macromolecules (Genheden & Ryde, 2015). With the help of these two techniques, it is possible to track dynamic changes in the protein-protein binding mode and assess such changes in terms of binding free energy.

In this study, we investigated a novel missense variant EDAR (c.1292 T>G; p.Ile431Arg) in a Chinese family with NSTA using whole-exome sequencing (WES) and Sanger sequencing methods. The pathogenic influence of the mutant is evaluated by bioinformatics analyses. Besides, we performed MD simulations and MM/GBSA binding free energy calculations to explore energetic and dynamic aspects of EDAR/EDARADD binding, which is important in the EDA/EDAR/NF-κB pathway.