Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

Salivary gland myoepithelial cells regulate saliva secretion and have been implicated in the histological diversity of salivary gland tumors. However, detailed functional analysis of myoepithelial cells has not been determined owing to the few of the specific marker to isolate them. We isolated myoepithelial cells from the submandibular glands of adult mice using the epithelial marker EpCAM and the cell adhesion molecule CD49f as indicators and found predominant expression of the transcription factor FoxO1 in these cells. RNA-sequence analysis revealed that the expression of cell cycle regulators was negatively regulated in FoxO1-overexpressing cells. Chromatin immunoprecipitation analysis showed that FoxO1 bound to the p21/p27 promoter DNA, indicating that FoxO1 suppresses cell proliferation through these factors. In addition, FoxO1 induced the expression of ectodysplasin A (Eda) and its receptor Eda2r, which are known to be associated with X-linked hypohidrotic ectodermal dysplasia and are involved in salivary gland development in myoepithelial cells. FoxO1 inhibitors suppressed Eda/Eda2r expression and salivary gland development in primordial organ cultures after mesenchymal removal. Although mesenchymal cells are considered a source of Eda, myoepithelial cells might be one of the resources of Eda. These results suggest that FoxO1 regulates myoepithelial cell proliferation and Eda secretion during salivary gland development in myoepithelial cells.

A missense mutation in the highly conserved TNF-like domain of Ectodysplasin A is the candidate causative variant for X-linked hypohidrotic ectodermal dysplasia in Limousin cattle: Clinical, histological, and molecular analyses.

Ectodysplasin A related hypohidrotic ectodermal dysplasia (XLHED) is a well-studied fetal developmental disorder in mammals that mainly affects ectodermal structures. It has been identified in a variety of species, including mice, rats, dogs, cattle, and humans. Here, we report the clinical, histological, and molecular biological analyses of a case of XLHED in Limousin cattle. An affected Limousin calf showed pathognomonic signs of ectodermal dysplasia, i.e. sparse hair and characteristic dental aplasia. Histopathologic comparison of hairy and glabrous skin and computed tomography of the mandible confirmed the phenotypic diagnosis. In addition, a keratoconjunctivitis sicca was noted in one eye, which was also confirmed histopathologically. To identify the causative variant, we resequenced the bovine X-chromosomal ectodysplasin A gene (EDA) of the affected calf and compared the sequences to the bovine reference genome. A single missense variant (rs439722471) at position X:g.80411716T>C (ARS-UCD1.3) was identified. The variant resulted in an amino acid substitution from glutamic acid to glycine within the highly conserved TNF-like domain. To rule out the possibility that the variant was relatively common in the cattle population we genotyped 2,016 individuals including 40% Limousin cattle by fluorescence resonance energy transfer analysis. We also tested 5,116 multibreed samples from Run9 of the 1000 Bull Genomes Project for the said variant. The variant was not detected in any of the cattle tested, confirming the assumption that it was the causative variant. This is the first report of Ectodysplasin A related hypohidrotic ectodermal dysplasia in Limousin cattle and the description of a novel causal variant in cattle.

A Causal Treatment for X-Linked Hypohidrotic Ectodermal Dysplasia: Long-Term Results of Short-Term Perinatal Ectodysplasin A1 Replacement.

X-linked hypohidrotic ectodermal dysplasia (XLHED), caused by a genetic deficiency of ectodysplasin A1 (EDA1), is a rare developmental disorder of ectodermal derivatives such as hair, sweat glands, and teeth. The absence of sweat glands and perspiration can evoke life-threatening hyperthermia. As molecular genetic findings are not always conclusive, the concentrations of circulating EDA1 may help to distinguish between total and partial EDA1 deficiencies. We previously treated nine male patients with obvious signs of XLHED with a recombinant EDA1 replacement protein, Fc-EDA, either shortly after birth (n = 3) or by prenatal administration in gestational week 26 and beyond (n = 6). Here, we present the long-term follow-up for up to six years. In patients who had received Fc-EDA after birth, neither sweat glands nor sweating ability were detected at the age of 12-60 months. In contrast, prenatal EDA1 replacement resulted in ample sweat gland development and pilocarpine-inducible sweating in all treated subjects, who also attained more permanent teeth than their untreated affected relatives. Normal perspiration has persisted for six years in the two oldest boys treated repeatedly with Fc-EDA in utero. When they had a sauna, adequate thermoregulation was evidenced. Lower sweat production after single prenatal dosing may indicate a dose-response relationship. The absence of circulating EDA1 in five prenatally treated subjects proved that these children would have been unable to perspire if they had been left untreated. The sixth infant was shown to produce an EDA1 molecule that, albeit interacting with its cognate receptor, cannot activate EDA1 signaling. In conclusion, a causal treatment of XLHED before birth is feasible.

A novel deletion of exon 4 in the Ectodysplasin A gene associated with X-linked hypohidrotic ectodermal dysplasia.

OBJECTIVE: Identify the disease-causing mutation in a patient with features of X-linked hypohidrotic ectodermal dysplasia, which is a genetic disorder characterized by hypodontia, hypohidrosis and hypotrichosis. It is caused by mutations in Ectodysplasin A gene, which encodes ectodysplasin A, a member of the tumor necrosis factor superfamily. DESIGN: Genetic analysis, was performed using chromosomal microarray analysis, whole exome sequencing and multiplex ligation-dependent probe amplification analysis in a 4-year-old boy with hypohidrotic ectodermal dysplasia features. Moreover, the boy's parents were tested for clinically significant findings identified in order to elucidate the pattern of inheritance of the finding detected in the proband. RESULTS: A novel deletion of entire exon 4 in Ectodysplasin A gene identified in the 4-year-old patient. This deletion was found in heterozygous state in the mother of the proband and was not detected in his father. RNA analysis revealed an in-frame deletion r.527_706del, p.(176_236del) in exon 4 of the Ectodysplasin A gene. CONCLUSION: We identified a novel gross deletion in the Ectodysplasin A gene in a male patient with X-linked hypohidrotic ectodermal dysplasia. Clinical and molecular genetic analysis are crucial to set an accurate diagnosis in patients with hypohidrotic ectodermal dysplasia. These results highlight the importance of the collagen domain of Ectodysplasin A, encoded by exon 4, for its function in vivo.

Next generation sequencing panel target genes: possible diagnostic tool for ectodermal dysplasia related diseases.

BACKGROUND: Ectodermal dysplasias (EDs) are a large and complex group of disorders affecting the ectoderm-derived organs; the clinical and genetic heterogeneity of these conditions renders an accurate diagnosis more challenging. The aim of this study is to demonstrate the clinical utility of a targeted resequencing panel through enhancing the molecular and clinical diagnosis of EDs. Given the recent developments in gene and protein-based therapies for X-linked hypohidrotic ectodermal dysplasia, there is a re-emerging interest in identifying the genetic basis of EDs and the respective phenotypic presentations, in an aim to facilitate potential treatments for affected families. METHODS: We assessed seventeen individuals, from three unrelated families, who presented with diverse phenotypes suggestive of ED. An extensive multidisciplinary clinical evaluation was performed followed by a targeted exome resequencing panel (including genes that are known to cause EDs). MiSeqTM data software was used, variants with Qscore >30 were accepted. RESULTS: Three different previously reported hemizygous EDA mutations were found in the families. However, a complete genotype-phenotype correlation could not be established, neither in our patients nor in the previously reported patients. CONCLUSIONS: Targeted exome resequencing can provide a rapid and accurate diagnosis of EDs, while further contributing to the existing ED genetic data. Moreover, the identification of the disease-causing mutation in an affected family is crucial for proper genetic counseling and the establishment of a genotype-phenotype correlation which will subsequently provide the affected individuals with a more suitable treatment plan.

Structural insights into pathogenic mechanism of hypohidrotic ectodermal dysplasia caused by ectodysplasin A variants.

EDA is a tumor necrosis factor (TNF) family member, which functions together with its cognate receptor EDAR during ectodermal organ development. Mutations of EDA have long been known to cause X-linked hypohidrotic dysplasia in humans characterized by primary defects in teeth, hair and sweat glands. However, the structural information of EDA interaction with EDAR is lacking and the pathogenic mechanism of EDA variants is poorly understood. Here, we report the crystal structure of EDA C-terminal TNF homology domain bound to the N-terminal cysteine-rich domains of EDAR. Together with biochemical, cellular and mouse genetic studies, we show that different EDA mutations lead to varying degrees of ectodermal developmental defects in mice, which is consistent with the clinical observations on human patients. Our work extends the understanding of the EDA signaling mechanism, and provides important insights into the molecular pathogenesis of disease-causing EDA variants.

Ectodysplasin Signaling through XEDAR Is Required for Mammary Gland Morphogenesis.

XEDAR is a member of the TNF receptor subfamily and a mediator of the ectodysplasin (EDA) pathway. EDA signaling plays evolutionarily conserved roles in the development of the ectodermal appendage organ class, which includes hair, eccrine sweat glands, and mammary glands. Loss-of-function sequence variants of EDA, which encodes the two major ligand isoforms, EDA-A1 and EDA-A2, result in X-linked hypohidrotic ectodermal dysplasia characterized by defects in two or more types of ectodermal appendages. EDA-A1 and EDA-A2 signal through the receptors EDAR and XEDAR, respectively. Although the contributions of the EDA-A1/EDAR signaling pathway to EDA-dependent ectodermal appendage phenotypes have been extensively characterized, the significance of the EDA-A2/XEDAR branch of the pathway has remained obscure. In this study, we report the phenotypic consequences of disrupting the EDA-A2/XEDAR pathway on mammary gland differentiation and growth. Using a mouse Xedar knockout model, we show that Xedar has a specific and temporally restricted role in promoting late pubertal growth and branching of the mammary epithelium that can be influenced by genetic background. Our findings implicate Xedar in ectodermal appendage development and suggest that the EDA-A2/XEDAR signaling axis contributes to the etiology of EDA-dependent mammary phenotypes.

Protocol for the Phase 2 EDELIFE Trial Investigating the Efficacy and Safety of Intra-Amniotic ER004 Administration to Male Subjects with X-Linked Hypohidrotic Ectodermal Dysplasia.

X-linked hypohidrotic ectodermal dysplasia (XLHED) is a rare genetic disorder characte-rised by abnormal development of the skin and its appendages, such as hair and sweat glands, the teeth, and mucous glands of the airways, resulting in serious, sometimes life-threatening complications like hyperthermia or recurrent respiratory infections. It is caused by pathogenic variants of the ectodysplasin A gene (EDA). Most affected males are hemizygous for EDA null mutations that lead to the absence or inactivity of the signalling protein ectodysplasin A1 (EDA1) and, thus, to the full-blown phenotype with inability to perspire and few if any teeth. There are currently no long-term treatment options for XLHED. ER004 represents a first-in-class protein replacement molecule designed for specific, high-affinity binding to the endogenous EDA1 receptor (EDAR). Its proposed mechanism of action is the replacement of missing EDA1 in yet unborn patients with XLHED. Once bound to EDAR, ER004 activates the EDA/NFκB signalling pathway, which triggers the transcription of genes involved in the normal development of multiple tissues. Following preclinical studies, named-patient use cases demonstrated significant potential of ER004 in affected males treated in utero during the late second and third trimesters of pregnancy. In order to confirm these results, we started the EDELIFE trial, a prospective, open-label, genotype-match controlled, multicentre clinical study to investigate the efficacy and safety of intra-amniotic ER004 administration as a prenatal treatment for male subjects with XLHED. This article summarises the rationale, the study protocol, ethical issues of the trial, and potential pitfalls.

A novel mutation in the collagen domain of EDA results in hypohidrotic ectodermal dysplasia by impacting the receptor-binding capability.

BACKGROUND: Hypohidrotic ectodermal dysplasia (HED) mainly results from gene mutations in the EDA/EDAR/NF-κB pathway. Function analysis of the mutations in the collagen domain of ectodysplasin A (EDA)result in HED has been rarely studied. This study aimed at determining the mechanism by which the novel collagen domain mutation of EDA results in HED. METHODS: We analyzed the DNAs from a Chinese family with HED and performed bioinformatics analysis. A new three-dimensional structure model of the EDA trimer was built and used to predict the effect of the mutations on EDA. We performed a western blot to detect EDA1 proteins in cell lysates and supernatants. We then performed coimmunoprecipitation to determine whether the mutation would affect the interaction of EDA1 with the EDA receptor (EDAR). Dual luciferase reporter assay and immunofluorescence were performed to detect the effect of the mutant EDA1 protein on nuclear factor kappa B (NF-κB) activation. RESULTS: A novel missense mutation (c.593G > A, p. Gly198Glu) in the collagen domain of EDA was detected. The mutation was predicted to be disease-causing. A three-dimensional structure model of the EDA trimer was first built in this study, in which the mutation site is located around the receptor binding domain. Functional studies showed that there was no difference in the secretion activity between the mutant EDA1 and the wild-type EDA1. However, the receptor-binding activity and the transcription activation of NF-κB were impaired by the mutation. CONCLUSION: We identified a novel mutation (c.593G > A, p. Gly198Glu) in the collagen domain of EDA. Bioinformatics analysis and functional studies showed this mutation was damaging, indicating that mutations in the collagen domain of EDA could result in HED by affecting the receptor-binding activity of EDA and the transcriptional activity of NF-κB.

A novel EDAR variant identified in non-syndromic tooth agenesis: Insights from molecular dynamics.

OBJECTIVE: This study aims to investigate a novel pathogenic variant in a Chinese family of non-syndromic tooth agenesis (NSTA) and study the impact of the variant on related protein and pathway. DESIGN: One NSTA family was collected. Whole exome sequencing and Sanger sequencing were performed on the proband with NSTA and his 5 family members. The pathogenic influence of the mutant is evaluated by bioinformatics analyses including evolutionary conservation analysis and secondary structure prediction. Molecular dynamics (MD) simulations and binding free energy calculations were then performed to explore changes in the tertiary structure and binding ability of the protein. RESULTS: We found a novel missense ectodysplasin A receptor (EDAR) variant (c .1292 T > G; p.Ile431Arg) in all affected family members. The results of bioinformatics analyses revealed that the EDAR had harmful changes after mutation. MD simulations and the binding free energy calculations results showed that the mutant EDAR protein and EDAR/ectodysplasin-A receptor-associated adapter (EDARADD) complex displayed tertiary structural change, and EDAR possessed a lower affinity to EDARADD after mutation. CONCLUSIONS: We found a novel EDAR variant (c.1292 T > G; p.Ile431Arg) in one NSTA family, which affects the binding of EDAR and EDARADD.

Ectodysplasin A1 Deficiency Leads to Osteopetrosis-like Changes in Bones of the Skull Associated with Diminished Osteoclastic Activity.

Pathogenic variants of the gene Eda cause X-linked hypohidrotic ectodermal dysplasia (XLHED), which is characterized by structural abnormalities or lack of ectodermal appendages. Signs of dysplasia are not restricted to derivatives of the ectodermal layer, but mesodermal abnormalities, such as craniofacial dysmorphism, are also frequently observed, suggesting close reciprocal interactions between the ectoderm and mesoderm; however, a causal link has remained unsubstantiated. We investigated the functional impact of defective ectodysplasin A1 (Eda1) signaling on postnatal bone homeostasis in Eda1-deficient Tabby mice. Interestingly, Eda1 was detected in wild-type mouse calvariae throughout postnatal lifetime. In calvariae, bone-lining Osterix (Osx)+ osteoblasts stained positive for Eda1, and osteoclasts were revealed as Eda receptor (Edar)-positive. Moreover, adult Eda1-deficient calvarial bone showed osteopetrosis-like changes with significantly diminished marrow space, which was maintained during adulthood. Concomitantly with osteopetrosis-like changes, Tabby calvarial bone and Tabby bone marrow-derived osteoclasts had far less osteoclastic activity-associated co-enzymes including cathepsin K, Mmp9, Trap, and Tcirg1 (V-type proton ATPase a3 subunit) compared with wild-type calvariae in vivo or osteoclasts in vitro, indicating that Eda1 deficiency may affect the activity of osteoclasts. Finally, we confirmed that nuclear Nfatc1-positive osteoclasts were strongly diminished during mature osteoclastic differentiation under M-CSF and RANKL in the Tabby model, while Fc-EDA treatment of Tabby-derived osteoclasts significantly increased nuclear translocation of Nfatc1. Furthermore, we identified enhanced Nfatc1 and NF-κB transcriptional activity following Fc-EDA treatment in vitro using luciferase assays. Overall, the results indicate that diminished expressions of osteoclastic activity-associated co-enzymes may lead to disturbed bone homeostasis in Tabby calvariae postnatally.

A large deletion encompassing exon 2 of the ectodysplasin A (EDA) gene in a British blue crossbred calf with hypohidrotic ectodermal dysplasia.

BACKGROUND: Hypohidrotic ectodermal dysplasia (HED) is a congenital syndrome of mammals affecting organs and tissues of ectodermal origin characterized by absence or hypoplasia of hair, teeth, and eccrine glands. The disorder has been reported in several species, including humans, mice, dogs and cattle, associated with variants in genes affecting the ectodysplasin pathway, including the X-linked ectodysplasin A (EDA) gene. Until now, nine pathogenic variants have been found in the bovine EDA gene. Here we report a novel variant in EDA in a crossbreed male Belgian Blue calf with HED, and provide an overview of the phenotypic and allelic heterogeneity of EDA-related forms of HED in cattle. CASE PRESENTATION: A 45-day-old male crossbreed British Blue calf was referred with congenital hypotrichosis, oligodontia and omphalitis. On histopathological examination of the nasal planum, nasolabial glands and ducts were not observed. The density of hair follicles was low, and they were small, with a predominance of telogen-phase hairs, and some serocellular crusts. The phenotype of the calf resembled that of HED. Whole-genome sequencing (WGS) was performed and revealed a 21,899 base-pair deletion encompassing the coding exon 2 of EDA, predicted to result in an altered transcript and aberrant protein. CONCLUSIONS: The clinicopathological and genetic findings were consistent with a case of X-linked HED. A very similar EDA deletion has been previously reported in a family of Holstein cattle with HED. The newly identified hemizygous EDA loss-of-function variant is certainly pathogenic and therefore is the genetic cause for the observed phenotype. This case report provides an additional example of the potential of WGS-based precise diagnostics in livestock species such as cattle to increase the diagnostic yield in rare diseases.

Ectodysplasin A (EDA) Signaling: From Skin Appendage to Multiple Diseases.

Ectodysplasin A (EDA) signaling is initially identified as morphogenic signaling regulating the formation of skin appendages including teeth, hair follicles, exocrine glands in mammals, feathers in birds and scales in fish. Gene mutation in EDA signaling causes hypohidrotic ectodermal dysplasia (HED), a congenital hereditary disease with malformation of skin appendages. Interestingly, emerging evidence suggests that EDA and its receptors can modulate the proliferation, apoptosis, differentiation and migration of cancer cells, and thus may regulate tumorigenesis and cancer progression. More recently, as a newly discovered hepatocyte factor, EDA pathway has been demonstrated to be involved in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and type II diabetes by regulating glucose and lipid metabolism. In this review, we summarize the function of EDA signaling from skin appendage development to multiple other diseases, and discuss the clinical application of recombinant EDA protein as well as other potential targets for disease intervention.

Understanding the impact of missense mutations on the structure and function of the EDA gene in X-linked hypohidrotic ectodermal dysplasia: A bioinformatics approach.

X-linked hypohidrotic dysplasia (XLHED), caused by mutations in the EDA gene, is a rare genetic disease that affects the development and function of the teeth, hair, nails, and sweat glands. The structural and functional consequences of caused by an ectodysplasin-A (EDA) mutations on protein phenotype, stability, and posttranslational modifications (PTMs) have not been well investigated. The present investigation involves five missense mutations that cause XLHED (L56P, R155C, P220L, V251M, and V322A) in different domains of EDA (TM, furin, collagen, and tumor necrosis factor [TNF]) from previously published papers. The deleterious nature of EDA mutant variants was identified using several computational algorithm tools. The point mutations induce major drifts in the structural flexibility of EDA mutant variants and have a negative impact on their stability, according to the 3D protein modeling tool assay. Using the molecular docking technique, EDA/EDA variants were docked to 10 EDA interacting partners, retrieved from the STRING database. We found a novel biomarker CD68 by molecular docking analysis, suggesting all five EDA variants had lower affinity for EDAR, EDA2R, and CD68, implying that they would affect embryonic signaling between the ectodermal and mesodermal cell layers. In silico research such as gene ontology, subcellular localization, protein-protein interaction, and PTMs investigations indicates major functional alterations would occur in EDA variants. According to molecular simulations, EDA variants influence the structural conformation, compactness, stiffness, and function of the EDA protein. Further studies on cell line and animal models might be useful in determining their specific roles in functional annotations.

Ectodysplasin pathogenic variants affecting the furin-cleavage site and unusual clinical features define X-linked hypohidrotic ectodermal dysplasia in India.

Hypohidrotic ectodermal dysplasia (HED) is a rare genetic disorder caused by mutational inactivation of a developmental pathway responsible for generation of tissues of ectodermal origin. The X-linked form accounts for the majority of HED cases and is caused by Ectodysplasin (EDA) pathogenic variants. We performed a combined analysis of 29 X-linked hypohidrotic ectodermal dysplasia (XLHED) families (including 12 from our previous studies). In addition to the classical triad of symptoms including loss (or reduction) of ectodermal structures, such as hair, teeth, and sweat glands, we detected additional HED-related clinical features including facial dysmorphism and hyperpigmentation in several patients. Interestingly, global developmental delay was identified as an unusual clinical symptom in many patients. More importantly, we identified 22 causal pathogenic variants that included 15 missense, four small in-dels, and one nonsense, splice site, and large deletion each. Interestingly, we detected 12 unique (India-specific) pathogenic variants. Of the 29 XLHED families analyzed, 11 (38%) harbored pathogenic variant localized to the furin cleavage site. A comparison with HGMD revealed significant differences in the frequency of missense pathogenic variants; involvement of specific exons and/or protein domains and transition/transversion ratios. A significantly higher proportion of missense pathogenic variants (33%) localized to the EDA furin cleavage when compared to HGMD (7%), of which p.R155C, p.R156C, and p.R156H were detected in three families each. Therefore, the first comprehensive analysis of XLHED from India has revealed several unique features including unusual clinical symptoms and high frequency of furin cleavage site pathogenic variants.

Association of EDARV370A with breast density and metabolic syndrome in Latinos.

The ectodysplasin receptor (EDAR) is a tumor necrosis factor receptor (TNF) superfamily member. A substitution in an exon of EDAR at position 370 (EDARV370A) creates a gain of function mutant present at high frequencies in Asian and Indigenous American populations but absent in others. Its frequency is intermediate in populations of Mexican ancestry. EDAR regulates the development of ectodermal tissues, including mammary ducts. Obesity and type 2 diabetes mellitus are prevalent in people with Indigenous and Latino ancestry. Latino patients also have altered prevalence and presentation of breast cancer. It is unknown whether EDARV370A might connect these phenomena. The goals of this study were to determine 1) whether EDARV370A is associated with metabolic phenotypes and 2) if there is altered breast anatomy in women carrying EDARV370A. Participants were from two Latino cohorts, the Arizona Insulin Resistance (AIR) registry and Sangre por Salud (SPS) biobank. The frequency of EDARV370A was 47% in the Latino cohorts. In the AIR registry, carriers of EDARV370A (GG homozygous) had significantly (p < 0.05) higher plasma triglycerides, VLDL, ALT, 2-hour post-challenge glucose, and a higher prevalence of prediabetes/diabetes. In a subset of the AIR registry, serum levels of ectodysplasin A2 (EDA-A2) also were associated with HbA1c and prediabetes (p < 0.05). For the SPS biobank, participants that were carriers of EDARV370A had lower breast density and higher HbA1c (both p < 0.05). The significant associations with measures of glycemia remained when the cohorts were combined. We conclude that EDARV370A is associated with characteristics of the metabolic syndrome and breast density in Latinos.

Gene Mutations of the Three Ectodysplasin Pathway Key Players (EDA, EDAR, and EDARADD) Account for More than 60% of Egyptian Ectodermal Dysplasia: A Report of Seven Novel Mutations.

Ectodermal dysplasia (ED) is a diverse group of genetic disorders caused by congenital defects of two or more ectodermal-derived body structures, namely, hair, teeth, nails, and some glands, e.g., sweat glands. Molecular pathogenesis of ED involves mutations of genes encoding key proteins of major developmental pathways, including ectodysplasin (EDA) and wingless-type (WNT) pathways. The most common ED phenotype is hypohidrotic/anhidrotic ectodermal dysplasia (HED) featuring hypotrichosis, hypohidrosis/anhidrosis, and hypodontia. Molecular diagnosis is fundamental for disease management and emerging treatments. We used targeted next generation sequencing to study EDA, EDAR, EDARADD, and WNT10A genes in 45 Egyptian ED patients with or without hypohidrosis. We present genotype and phenotype data of 28 molecularly-characterized patients demonstrating genetic heterogeneity, variable expressivity, and intrafamilial phenotypic variability. Thirteen mutations were reported, including four novel EDA mutations, two novel EDARADD, and one novel EDAR mutations. Identified mutations congregated in exons encoding key functional domains. EDA is the most common gene contributing to 85% of the identified Egyptian ED genetic spectrum, followed by EDARADD (10%) and EDAR (5%). Our cohort represents the first and largest cohort from North Africa where more than 60% of ED patients were identified emphasizing the need for exome sequencing to explore unidentified cases.