Functional analysis of an Ectodysplasin mutation causing isolated tooth agenesis

Congenital, non-syndromic tooth agenesis (TA) is one of the most common anomalies of tooth development. Our current understanding implicates mutations in PAX9, MSX1, AXIN2 and EDA in its pathogenesis. Mutations in EDA are associated with X-linked hereditary ectodermal dysplasia.

Objective: To determine the functional consequences of a recently identified missense mutation (V365A) causing an X-linked recessive form of non-syndromic tooth agenesis.

Methods: Site-directed mutagenesis was used to introduce the V365A mutation into two pCR3 vectors encoding two splice forms of EDA: EDA-A1, which contains the TNF region and the other a six base-pair shorter region, EDA-A2. Vectors containing mutant and wild-type (wt) inserts were transfected into 293T cells. Cell extracts and supernatants were investigated by Western blot for presence of EDA expression products. Qualitative and quantitative receptor binding assays were performed using recombinant EDAR and XEDAR derived receptor proteins and recombinant EDA proteins from cell supernatants. The co-precipitated ligand was detected by Western blot. For quantitative receptor binding, ELISA was performed with quantified EDA ligand and one of two receptors (EDAR-Fc or XEDAR-Fc).

Results: Both EDA-A1 V365A and EDA-A2 V365A are expressed and glycosylated. Both are secreted similar to wt proteins. EDA-A1 V365A, like wt EDA-A1, co-precipitates with its receptor EDAR-Fc and EDA-A2 V365A co-precipitates with XEDAR-Fc. Quantitative ELISA showed that the affinity of EDA-A1 V365A to its receptor EDAR is reduced compared to wt. The affinity of the V365A EDA-A2 construct to XEDAR is similar to or even better than wt.

Conclusions: The V365A mutation only weakens ligand-receptor binding of EDA to its receptor EDAR, while other EDA mutations causing XL-HED that were previously tested abolish receptor binding. These findings suggest that the development of teeth requires the highest dosage of EDA signaling, while the other ectodermal appendages appear to be less sensitive to suboptimal receptor activation.