Conservation analysis and pathogenicity prediction of mutant genes of ectodysplasin a

Hypohidrotic ectodermal dysplasia (HED), also known as congenital ectodermal dysplasia syndrome, is a group of common hereditary diseases that affect the development of ectoderm. It often affects the development of more than two ectoderm-derived organs. The prevalence is approximately 1/100000 [1]. Clinical manifestations of HED mainly include hypotrichosis, loss or maldevelopment of sweat glands and nail dysplasia [2]. The main oral symptoms are alveolar bone dysplasia, craniofacial deformity and loss of intermaxillary distance [3, 4]. A majority of patients present with congenital tooth dysplasia, small incisors or canine teeth in cone shape and even full-mouth anodontia [5, 6].

Most HED cases have an X recessive inheritance [7, 8] and their pathogenic gene is the EDA gene [9]. Approximately 95% of the total HED patients are induced by gene mutations such as nonsense mutation, missense mutation and small fragment deletion [10]. Like most recessive X-linked hereditary diseases, the HED pedigree members are primarily male, whereas the female carriers merely present with mild clinical manifestations [11]. About 30% of the female carriers are asymptomatic [12].

In a preliminary study carried out by authors of the current study, peripheral blood of 4 unrelated HED pedigrees was collected. The peripheral blood of 100 healthy adults who don’t have the family history of congenitally missing tooth was used for comparison. The 4 symptomatic patients were subjected to a thorough clinical examination. Four male probands had typical clinical manifestations which were in line with X-linked recessive inheritance. The gene sequencing results demonstrated that the mutation was only found in the symptomatic patients and had patient to patient variability [13], including two point mutations (c.466C > T, c.878 T > G) and two deletion mutations (c.663-697del, C.587-615del). When compared with the reported gene mutation sites, point mutation (c.466C > T) in one pedigree was identified as a pathogenic mutation. The same is reported by Monreal 1988 [14]. The other three mutation sites (c.878 T > G, c.663-697del and c.587-615del) were new unidentified mutations.

SNP (single-nucleotide polymorphism) is the most common genome mutation. There are about a million SNP loci among humans. The SNP that is located in the gene coding region and can change the amino acid sequence leading to a missense mutation. With the improvement in disease diagnosis, there is a higher possibility of discovering new missense mutations [15]. In current times, researchers tend to use the different kinds of bioinformatics software to make the preliminary forecasting of the pathogenicity of the new mutation, recognize the related risks of the disease and offer the theoretical foundation using the cell model and the mouse disease model. In this study, conservation analysis of the related homologous proteins in 3 unknown EDA gene mutation sites was conducted by referring to the UCSC Genome Browser database. The online gene function prediction software SIFT and PolyPhen-2 were adopted to predict the pathogenicity of point mutation of c.878 T>. The aim of the study is to offer a reference for elucidating the pathogenesis of HED.

EDA gene is a causative gene of HED, which is located in the Xq12-q13 region. It contains 9 exons and encodes 391 amino acids. The encoded EDA protein is a type II trans-membrane protein with a short intracellular domain at the N-terminal and a TNF homology domain with 19 glycine residual repetitive sequence at the C-terminus [16]. It has multiple subtypes and mainly mediates epidermal-mesenchymal and cell-cell signaling transduction [17]. As an important member of tumor necrosis factor ligand family, EDA protein has been extensively studied in the field of developmental biology. It can regulate body morphogenesis and maintain the development of ectoderm-derived organs (tooth, hair, sweat gland and other cutaneous appendages) [18, 19]. Previous studies have demonstrated that application of the EDA gene-transfected bone mesenchymal stem cells can significantly improve the regenerative capacity of sweat glands and accelerate the potential repair and regeneration of the injured skin and its appendages [20]. However, after the EDA gene-inhibiting antibodies were implanted in wild-type pregnant mice, the wild-type mouse fetuses were found to have permanent ectodermal dysplasia [21].

EDA gene mutations are mainly located in four important functional areas [22]. ① The junction (TM) between the trans-membrane and extra-cellular parts which is related to the polarity of the amino acids. ② Furin cleavage is associated with the production of soluble molecules. The activity can be activated by the release of the soluble form of EDA proteins.③ Collagen-like domain (collagen) can bind two or more EDA trimer together, thereby stimulating the downstream signaling pathway.④ TNF homology domain (THD) can affect the formation of trimer and its specific binding with receptors. More than 100 types of EDA mutations have been found to have heterogeneity and ethnic differences [23, 24]. It is reported that 95% of the gene mutations occur in the 1, 3, 5, 8 and 9 exons, including missense mutation, small fragment insertion or deletion mutation and nonsense mutation [25, 26]. In addition, the deletion or translocation of single exon or full-length gene fragments has also been reported [27].

Mutations in the EDA gene can not only lead to congenital anodontia syndrome, but also provoke congenital anodontia with relatively mild symptoms, which are not complicated with the developmental defects of ectoderm-derived tissues and organs [26]. Tao et al. have reported of a Mongolian family with congenital anodontia. Besides congenital anodontia, no other clinical manifestations of HED were present in the affected family members. A new missense mutation (c.198C > G) was found in the EDA gene among all affected male and female carriers [28]. Song et al. [29] collected 15 unrelated male patients with asymptomatic congenital anodontia. Gene mutation testing revealed three novel mutation sites (p.Ala259Glu, p.Arg289Gys and p. Arg334His) in EDA gene of four patients with a detection rate of 27%. The genetic defects of EDA gene could lead to the incidence of asymptomatic congenital anodontia.

Along with the development of genetics and genomics fields, the EDA genes originally found in both patients and mouse models have been proven to contain a highly-conserved TNF homology domain in vertebrates and mammals [22, 30]. The findings in this study have demonstrated that the c.878 T > G mutation is located in the TNF homology domain of EDA gene. Mutations in this region can affect the binding of EDA protein to its receptors, resulting in the inactivation of NF-κB signaling pathway, thereby leading to the developmental defects of ectoderm-derived tissues and organs. NF-κB signaling pathway is now recognized as a classical signaling pathway associated with the incidence of HED [31]. C.878 T > G family is a large clinical pedigree. The nephews and cousins of the probands shared similar manifestations. Genetic testing showed that the base 878 of exon 7 was mutated from thymine to guanine in EDA gene of the probands and their nephews, and thus the CTG code was mutated into CGG, resulting in a change from leucine to arginine at the amino acid 293 (p.Leu293Arg). Genotype testing of this mutation at the gene level is the cause of hypoplastic ectodermal hypoplasia in this pedigree. In addition, further examination was carried out using the prediction software SIFT and PolyPhen-2. The results indicated that the mutation site possessed strong pathogenicity theoretically. The databases of 1000 Genome Project, the NHLBI GO Exome Sequencing Project and dbSNP database, were used to identify the frequency of the mutations. This further supported the pathogenicity of this mutation. However, the specific mechanism is not well understood. The study for the new missense mutation (c.878 T > G) aims to offer the theoretical support for the next functional experiment. Next, our research group will transfect the eukaryotic cell that is related to tooth development, and make a evaluate the effects of mutated EDA genes on tissues and organs of ectodermal origin at the protein level with a goal of guiding the clinical treatment.

The other two mutations (c.663-697del, c.587-615del), which have not been reported, are located in the collagen-like domain of EDA gene. These mutations lead to the frame-shifting mutation of exon 4 and induce a termination codon in advance at the 226th and 238th amino acids, resulting in a truncated protein. This causes functional defects of the EDA protein, which is unable to form normal EDA trimer, weakens the activation of downstream signaling pathways and affects the development of ectoderm-derived tissues and organs. Due to the complexities involved in simulating computer algorithms, the numerical procedure to predict the functional effects of these two unknown deletion mutations was not used. This may be carried out further studies.

Three unknown mutation sites (c.878 T > G, c.663-697del and c.587-615del) from the pedigrees of HED possess strong conservation of amino acid among different species and the mutation site of c.878 T > G holds strong pathogenicity theoretically. This may be responsible for the pathogenesis of HED in their pedigrees and it can also provide a reference for the pathogenicity of a wide range prediction of gene mutations.