Ectodermal dysplasia is a genetically and phenotypically heterogeneous condition characterized by the abnormal development of ectodermal derived structures. Although there have been a few other genes reported in individuals with ED-related findings, for hypohidrotic ectodermal dysplasia (HED) four main genes, namely EDA (the most common), in addition to EDAR, WNT10A, and EDARADD, have been suggested to be causative [8,9,10,11]. Neonatologists and paediatricians should raise the suspicion of HED in the presence of congenital skin defects, keeping in mind that such disorder may also be linked with mutations different than those affecting the EDA gene, including EDARADD [12]. Moreover, both recessive and dominant forms of HED caused by EDARADD mutations are clinically indistinguishable, and therefore in these cases genetic investigations are decisive for diagnosis confirmation [3, 7, 13]. Indeed, our patients exhibited the triad of symptoms characteristic of HED, including very sparse and brittle hair, teeth anomalies and reduced sweating. Finally, clinicians have to perform a careful differential diagnosis, including conditions sustained by different genetic aetiology such as mutation in TP63 [14].

In our study, we conducted next-generation sequencing of EDARADD, and identified a homozygous variant c.322_323insCGGGC p.(Arg108ProfsTer7) causing a frameshift, and located 5’ upstream of the death domain in exon 6. Such variant has not been previously reported in HED individuals, and data from large population studies is insufficient to assess its frequency. The mutation is an insertion of 5 bases at position c.322, and is expected to impact the protein, made by 215 amino acids in its wild type, introducing a premature termination codon at position 108.

Further studies confirmed co-segregation of the variant in the parents, and identified the heterozygous state in a healthy brother. Based on the segregation analysis, we concluded that this variant causes the recessive form of the disease, as no other family member exhibited symptoms related to HED.

To date, ten different mutations in EDARADD have been associated with HED, with only three of these being deletion or insertion [4, 5, 7, 8, 15,16,17,18,19], and different possible molecular pathogenic mechanisms have been hypothesized (disruption of the interaction with EDAR; impairment of the wild-type EDARADD ability to activate NF-kB, disturbance of the multimerization of EDARADD). Additionally, a homozygous gross deletion c.131-?_189+?del was reported with a loss-of-function mechanism.

Zygosity-position-mutation type does not seem to consistently predict the severity of the condition. Published literature is not conclusive in regard to the pathogenic mechanism of EDARADD, probably due to very low number of reported variants. However, the loss-of-function mechanism has been considered as a causing process of the disease linked to EDARADD [16, 17]. This may be due to the loss of protein through nonsense mediated mRNA decay (NMD), leading to the production of truncated proteins that are missing the death domain in exon 6 (highly conserved among species, suggesting its essential function) [20].

Three previous reported variants causing premature termination codons, as shown in Table 1, are predicted to go NMD. Since the present variant is not predicted to go NMD but removes more than 10% of transcript, this might be the first truncating variant causing termination codon and not going NMD. Therefore, the predicted molecular pathogenic mechanism is the translation of mRNA containing a premature stop codon, and the production of truncated proteins that are missing the death domain in exon 6. The reported variant is located in the 5’ upstream sequence of the death domain (Fig. 1d), probably leading to its deficient or absent translation. Also, the phenotypic variability of the syndrome may be explained by different conditions such as modifier genetic variations, possible interactions with regulatory factors and/or the involvement of epigenetic mechanisms in addition to EDARADD pleiotropy, which has already been described in the literature regarding other genes [21, 22].

In conclusion, our study identified a novel homozygous insertion variant in EDARADD, causing autosomal recessive HED. The segregation analysis strongly supported the recessive inheritance model. Although further studies are needed, our report expands the understanding of the disease, highlighting how genetic testing plays a pivotal role in molecular diagnosis and family risk assessment, also in light of the clinical indistinguishability of the various forms of HED.