Functional studies for the TRAF6 mutation associated with hypohidrotic ectodermal dysplasia

Summary Background  Hypohidrotic ectodermal dysplasia (HED) is a rare condition characterized by hypotrichosis, hypohidrosis and hypodontia. A de novo heterozygous mutation in the tumour necrosis factor receptor‐associated factor 6 gene ( TRAF6 ) was recently identified in a patient with HED, while functional consequences resulting from the mutation remained unknown. Objectives  To determine the mechanism by which the TRAF6 mutation results in HED. Methods  We performed coimmunoprecipitation (co‐IP) studies to determine whether the mutation would affect the interaction of TRAF6 with transforming growth factor β‐activated kinase 1 (TAK1), TAK1‐binding protein 2 (TAB 2) and ectodysplasin‐A receptor‐associated death domain protein (EDARADD). We then performed co‐IP and glutathione S‐transferase‐pulldown assays to determine the TRAF6 binding sequences in EDARADD. In addition, we analysed the effect of the mutant TRAF6 protein on the affinity between wild‐type TRAF6 and EDARADD, as well as on EDARADD‐mediated nuclear factor (NF)‐κB activation. Results  The mutant TRAF6 protein was capable of forming a complex with TAK1 and TAB 2 in a similar way to wild‐type TRAF6. However, the mutant TRAF6 protein completely lost the affinity to EDARADD, while the wild‐type TRAF6 bound to the N‐terminal domain of EDARADD. Furthermore, the mutant TRAF6 inhibited the interaction between the wild‐type TRAF6 and EDARADD, and also potentially reduced the EDARADD‐mediated NF‐κB activity. Conclusions  We conclude that the mutant TRAF6 protein shows a dominant negative effect against the wild‐type TRAF6 protein, which is predicted to affect the EDARADD‐mediated activation of NF‐κB during the development of ectoderm‐derived organs, and to lead to the HED phenotype.