Anhidrotic ectodermal dysplasia (EDA), also called hypohidrotic ectodermal dysplasia (HED) is a genetic disease characterized by the absence or hypoplasia of hair, teeth, and eccrine sweat glands that has been reported in humans (OMIM305100), the tabby mouse mutants, cattle and dogs (Kere et al. 1996; Srivastava et al. 1997; Drögemüller et al. 2001; Casal et al. 2005). Although most cases of EDA display X-linked recessive inheritance, autosomal dominant and autosomal recessive forms also exist in humans (Shimomura et al. 2004; van der Hout et al. 2008). The EDA gene on the X chromosome encodes a protein, ectodysplasin-A (EDA), which is responsible for human X-linked EDA (Kere et al. 1996; Monreal et al. 1998). In 2001 a mutation on the bovine EDA gene in German Holstein cattle was also reported (Drögemüller et al. 2001). Here we describe a novel mutation of the EDA gene in which a 19-bp deletion in exon 1 in a male Holstein calves demonstrated the phenotypic features of EDA.
The affected Holstein male calf exhibited hairlessness on most of its entire body except for a part of the pinna and the distal parts of the extremities at birth. Lower jaw hypoplasia was observed, no incisors were evident, and only one molar was present on each side of the upper and lower jaws, respectively (Fig. 1a). Symptoms included depression, weakened suckling activity from birth, and involuntarily recumbency with respiratory distress eight days after birth. The dam of the affected calf had normal hair. And her primiparous male calf, a crossbreed between a Japanese Black cattle and a Holstein, was also affected with hairlessness over its entire body except for the inner part of the pinna. In this male maternal half-sib of the affected calf, chronic diarrhea was observed at approximately one month of age followed by death at two months after birth. Deliveries were normal and biochemical blood tests showed no specific abnormalities in either of the affected calves. Results for a sample from the dam subjected to a PCR test were negative for bovine viral diarrhea virus (BVDV) suggesting a possible cause of congenital hypotrichosis (Barlund et al. 2007). For the histopathologic examination, formalin-fixed paraffin-embedded sections of skin samples from affected calves stained with hematoxylin and eosin were used (Fig. 1b).
Mutation analyses of bovine EDA gene were performed by PCR amplification and sequencing of genomic DNA from the EDA affected calf, its dam and a control group of normal animals, by using of previously published primer sets (Drögemüller et al. 2001) and additional primers Exon3.1F (5′-TATGGACAGAGGAGCCTGGT-3′), Exon3.1R (5′-TGGGAAAATCTTCTGCAACC-3′), Exon9.1F (5′-GGTACTGCTCCCTTCACGAG-3′), Exon9.1R (5′-GGCAAAACGAGGTTGGAG-3′), Exon9.2R (5′-TACCTCATTCCACAGCAGCA-3′) and Exon1.2R (5′-GCCAAAGAAACCCAGAAAGA-3′). The PCR-amplified fragments of all eight exons and intronic exon-flanking regions were sequenced using the BigDye terminator chemistry ver. 3.1 (Applied Biosystems) on an ABI3700 sequencer (Applied Biosystems). Sequences were aligned and compared to reference sequences using DNASIS software ver. 2.06 (Hitachi Software). For screening this mutation, PCR amplification of genomic DNA using primers of Exon1.1F (5′-GCCTCAGAGAGTGGGTGTCT-3′) and Exon1.1R (5′-CGCAGTTCTAGGTAGCAGCA-3′) was performed and subsequent analysis by polyacrylamide gel electrophoresis (PAGE) was carried out (Fig. 2b).
Histopathologic examination indicated the presence of underdevelopment of hair follicles and adnexal glands (Fig. 1b). Direct sequencing of PCR-amplified DNA from the affected calf, its dam and normal animals showed that a 19-bp deletion at nucleotides c.48_66 in exon 1 leading to a frameshift results in a premature stop codon. This mutation is predicted to generate a truncated 49 aa protein. Except for exon 1, all other EDA exons were normal and showed no changes between the affected calf and normal animals. This deletion was hemizygous in the affected calf and heterozygous in its dam. Further analysis of this deletion from the grand-dam, grand-sire, male maternal half-sib of the affected calf, which was also characterized clinically as EDA, as well as from 34 normal sires and 248 unrelated normal dairy cows showed that the half-sib calf was hemizygous for this deletion, the grand-dam was heterozygous (Fig. 2), and other animals including the grand-sire were wild type. These results confirm that the family used in this study has the X-linked recessive form of EDA that is caused by the 19-bp deletion in exon 1 of the EDA gene on the X chromosome.
Previous studies showed that various mutations were present in the bovine EDA gene (Drögemüller et al 2001, 2002, 2003; Barlund et al. 2007). This study detected a novel mutation in the bovine EDA gene that lead to EDA in Holstein cattle. This mutation was a 19-bp deletion in exon 1 that predicted the generation of an aberrant truncated 49 aa protein as compared the with wild type that generates a 391 aa protein. Therefore, the mutation allele probably expresses a complete loss of function of the ectodysplasin-A protein that was encoded by the EDA gene. Ectodysplasin-A is a trimeric transmembrane protein that has a collagenous domain and a tumor necrosis factor-like signaling domain in the extracellular region. The extracellular region containing both domains was released from the cell membrane by cleavage of the ectodysplasin molecule by a furin-like protease. The collagenous domain mediates trimerization and the signaling domain induces receptor-mediated signal transduction through binds to the receptor. It is assumed to be involved in the early epithelial-mesenchymal interaction that controls the formation of fetal hair follicles and tooth buds. Many mutations in the human EDA gene and almost all mutations located in functional domains in the ectodysplasin-A protein were reported (Ferguson et al. 1997; Schneider et al. 2001).
With regard to EDA in canines, most affected dogs demonstrate the typical symptoms of EDA, including impaired development of hair, teeth, and sweat glands with a mildly compromised immune system. However, canine EDA is not always lethal. Casal et al. (1997) reported that it was possible to produce affected female dogs by mating affected male dogs with carrier female dogs experimentally. In humans, many patients show variable levels of severity. It is assumed that the affected calves in this study were samples of the most severe cases. It may be hypothesized that this severity is associated with the mutation allele detected in this study that demonstrates a complete loss of function of the all isoforms encoded by the EDA gene. Various splice forms of the EDA transcript have been detected (Bayés et al. 1998), especially two isoforms differing only by two amino acids, EDA-A1 (391 aa) and EDA-A2 (389 aa), binding to two different receptors, the EDA receptor (EDAR) and the X-linked EDA receptor (XEDAR), respectively (Yan et al. 2000; Schneider et al. 2001; Mikkola 2009). The biological significance of other isoforms remains unclear, but transcripts for all isoforms including EDA-A1 and EDA-A2 consist of exon 1 and other exons of the EDA gene. Therefore, it is assumed that the deletion close to the start codon confuses all transcripts, and results in the complete loss of pleiotropic functions of the bovine EDA gene. These results suggest that this mutation might serve as useful animal models for the investigation of the pathogenic mechanisms of the anhidrotic ectodermal dysplasia.