4 | DISCUSSION
Mutations of the EDAR gene can result in HED(A.K. et al., 2017; Feng et al., 2018). The Human Gene Mutation Database (HGMD Professional, 2018.3) has 62 registered pathogenic variants of EDAR , of which 50 have the HED phenotype(Parveen et al., 2019). In recent years, several EDAR mutations in NSTA patients have been identified(Arte et al., 2013; Jonsson et al., 2018; Mumtaz et al., 2020; Zeng et al., 2017). In our study, a newEDAR missense mutation c.338 G>A(Cys113Tyr) was detected by whole exome sequencing in Patient 4 and his mother. A cross-species alignment of amino acid sequences of EDAR showed that p.C113 was conserved across seven species, implying the high pathogenicity of the variant. Our result expands the mutation spectrum of EDAR .
EDAR is a type I transmembrane protein and a member of the TNF receptor superfamily. It has a cysteine-rich domain in the extracellular region (encoded by exons 3, 4 and 5), as well as a death domain in its intracellular region (encoded by exon 12). EDAR interacts with extracellular EDA1 and intracellular EDARADD via the extra- and intracellular regions to form a complex. This in turn activates downstream nuclear factor kappa B (NF-κB) to mediate transcription of the target gene(Kumar, Eby, Sinha, Jasmin, & Chaudhary, 2001; Masui et al., 2011; Okita, Asano, Yasuno, & Shimomura, 2019; Outi et al., 2001; Parveen et al., 2019). The p.Cys113Tyr (c.338G>A) mutation in exon 4 occurs in a region that encodes the cysteine-rich domain that binds to EDA1. We speculated that the p.Cys113Tyr substitution of EDAR might alter its affinity with EDA1, so that it instead binds to EDARADD, ultimately affecting the activation of downstream nuclear factor (NF)-κB.
By studying the information of all NSTA patients with EDARmutations, we found that more than half (5/8) of EDAR mutations in NSTA patients were concentrated in the death domain encoded by exon 12, and the majority of mutations (6/8) were missense mutations (Table 1). We also observed that the most common missing teeth were the maxillary lateral incisors, followed by the mandibular lateral incisors and the mandibular central incisors. The maxillary first premolars and mandibular canines were the least likely to be affected (Table 2; Figure 4). It seems that anterior teeth, especially the incisors, are sensitive to mutations in the EDAR gene. Arte et al. (Arte et al., 2013)and Mumtaz et al. (Mumtaz et al., 2020) found that both deciduous and permanent incisors were involved most commonly in those EDAR-related TA cases, is similar to our finding. In particular, the number of permanent teeth lost in patients with EDAR -related NSTA ranged from 2 to 10. In the present study, the proband and his mother had 18 and 17 permanent teeth missing, respectively. This differs from previous studies, but our participants presented with no signs of other ectodermal hypoplasias, such as in the hair or skin. We hypothesized that the differences in the number of missing teeth might be associated with single nucleotide polymorphisms or epigenetic factors.
According to M. Yu et al.(M. Yu et al., 2019), over the past two decades 198 different mutations had been detected that are responsible for NSTA, of which 27 are derived fromEDA1 . Previous studies revealed a clear link between the genotype and phenotype for congenital tooth deficiency(Han et al., 2008; He, Liu, Han, Liu, & Feng, 2016; Wong et al., 2018; Zhang et al., 2011). Han et al.(Han et al., 2008) studied 24 NSTA patients with EDA1 mutations and conducted statistical analysis on the number of missing teeth in each position of dentition. They found that the most likely missing teeth were the lateral incisors followed by the mandibular central incisors; and the least likely missing teeth were the maxillary central incisors and first permanent molars. The results of He et al.(He et al., 2016) also confirmed that these characteristics were specific phenotypes of NSTA caused by EDA1 mutations.
In our research, three mutations were found in the EDA1 gene of oligodontia Patient 1-3. The probands shared the EDA1 mutations with their mother (Figure 3), indicating that the mutant alleles were inherited from the maternal line in Family 1, 2, and 3. Two of the mutations (c.865C>T and c.866G>A) observed in this study were located in the TNF domain of EDA1 . The TNF homology domain forms a homotrimer, which is required for interaction with the receptor at the monomer–monomer interface(Hymowitz et al., 2000). Song et al.(Song et al., 2009) carried out structural analysis on the EDA1 protein and found that in the wild-type 3D EDA1 structure, the Arg289 residue is located at the outer surface of the homotrimer and makes structural hydrogen bonds with Asn272. In addition, Arg289 forms hydrogen bonds and electrostatic interactions with Glu308 of the adjacent homotrimer(s) to stabilize the multi-trimer asymmetric unit(Lee et al., 2014; Ruiz-Heiland et al., 2016; Song et al., 2009). Variants at the p.289 location would abolish these interactions and reduce protein stability. Therefore, codon 289 mutations in the TNF domain partly impact on the binding of EDA protein to EDA receptor.
However, it is worth noting that although all changes occurred at p.289 of EDA1 , the phenotypes of patients were slightly different. We performed a genotype-phenotype analysis and found that all maxillary lateral incisors are affected, followed by mandibular incisors and mandibular second premolars. In contrast, the second molars, mandibular first molars and maxillary central incisors were less affected. It is particularly interesting that the maxillary first molars were present in all patients. Our phenotypic analysis of the mutations at codon 289 ofEDA1 is consistent with the typical phenotype resulting fromEDA1 gene mutations. However, the mechanism that causes the slight changes to the phenotype of teeth loss due to protein mutations remains to be studied.