Mutation identification in a canine model of X-linked ectodermal dysplasia

Abstract

X-linked hypohidrotic ectodermal dysplasia (XHED), an inherited disease recognized in humans, mice, and cattle, is characterized by hypotrichosis, a reduced number or absence of sweat glands, and missing or malformed teeth. In a subset of affected individuals and animals, mutations in the EDA gene (formerly EDI), coding for ectodysplasin, have been found to cause this phenotype. Ectodysplasin is a homotrimeric transmembrane protein with an extracellular TNF-like domain, which has been shown to be involved in the morphogenesis of hair follicles and tooth buds during fetal development. Some human XHED patients also have concurrent immunodeficiency, due to mutations in the NF-kappaB essential modulator protein (IKBKG; formerly NEMO), which is also encoded on the X chromosome. In a breeding colony of dogs with XHED, immune system defects had been suspected because of frequent pulmonary infections and unexpected deaths resulting from pneumonia. To determine if defects in EDA or IKBKG cause XHED in the dogs, linkage analysis and sequencing experiments were performed. A polymorphic marker near the canine EDA gene showed significant linkage to XHED. The canine EDA gene was sequenced and a nucleotide substitution (G to A) in the splice acceptor site of intron 8 was detected in affected dogs. In the presence of the A residue, a cryptic acceptor site within exon 9 is used, leading to a frame shift and use of a premature stop codon that truncates the translation of both isoforms, EDA-A1 and EDA-A2, resulting in the absence of the TNF-like homology domain, the receptor-binding site of ectodysplasin.

Fig. 1

Percent similarity of cDNA and amino acid sequences of EDA from cattle, dogs, mice, and humans. Exons 7–9 are completely homologous among dogs, cattle, mice, and humans (not shown).

Fig. 2

(A) The homotrimeric protein encoded by EDA. The amino acid sequence expressed by exons 7–9 is 100% homologous in mice, dogs, cattle, and humans. IC: intracellular, TM: transmembrane, EC: extracellular domains. (B) Genomic organization of EDA in dogs and the effect of the mutation. A cryptic splice acceptor site within exon 9 is used in the affected dogs, resulting in a frame shift and a premature stop codon. The numbers above the figure indicate exon sizes and those below indicate the position within the exon.