Mutations leading to X-linked hypohidrotic ectodermal dysplasia affect three major functional domains in the tumor necrosis factor family member ectodysplasin-A

Mutations in the epithelial morphogen ectodysplasin-A (EDA), a member of the tumor necrosis factor (TNF) family, are responsible for the human disorder X-linked hypohidrotic ectodermal dysplasia (XLHED) characterized by impaired development of hair, eccrine sweat glands, and teeth. EDA-A1 and EDA-A2 are two splice variants of EDA, which bind distinct EDA-A1 and X-linked EDA-A2 receptors. We identified a series of novel EDA mutations in families with XLHED, allowing the identification of the following three functionally important regions in EDA: a C-terminal TNF homology domain, a collagen domain, and a furin protease recognition sequence. Mutations in the TNF homology domain impair binding of both splice variants to their receptors. Mutations in the collagen domain can inhibit multimerization of the TNF homology region, whereas those in the consensus furin recognition sequence prevent proteolytic cleavage of EDA. Finally, a mutation affecting an intron splice donor site is predicted to eliminate specifically the EDA-A1 but not the EDA-A2 splice variant. Thus a proteolytically processed, oligomeric form of EDA-A1 is required in vivo for proper morphogenesis.     

Mutation p.Leu354Pro in EDA causes severe hypohidrotic ectodermal dysplasia in a Chinese family

X-linked recessive hypohidrotic ectodermal dysplasia (XLHED) is characterized by the defective morphogenesis of teeth, hair, and eccrine sweat glands. It is associated with mutations in the EDA gene. Up to now, more than 100 mutations in the EDA gene have been reported to cause XLHED. The product of EDA gene is a trimeric type II transmembrane protein that belongs to the tumor necrosis factor (TNF) family of ligands. In this study, we identified a Chinese family with XLHED. Direct DNA sequencing of the whole coding region of EDA revealed a novel missense mutation, p.Leu354Pro in a patient affected with XLHED. This mutation was not found in either unaffected male individuals of the family or 168 normal controls. The substitution of Leu354 with Pro was found to be located in the TNF-like domain of EDA and may influence the epithelial signaling pathway required for the normal ectodermal development through altering the topology of EDA. Our finding broadens the spectrum of EDA mutations and may help to understand the molecular basis of XLHED and aid genetic counseling.     

Permanent correction of an inherited ectodermal dysplasia with recombinant EDA

X-linked hypohidrotic ectodermal dysplasia (XLHED; OMIM 305100) is a genetic disorder characterized by absence or deficient function of hair, teeth and sweat glands. Affected children may experience life-threatening high fever resulting from reduced ability to sweat. Mice with the Tabby phenotype share many symptoms with human XLHED patients because both phenotypes are caused by mutations of the syntenic ectodysplasin A gene (Eda) on the X chromosome. Two main splice variants of Eda, encoding EDA1 and EDA2, engage the tumor necrosis factor (TNF) family receptors EDAR and XEDAR, respectively. The EDA1 protein, acting through EDAR, is essential for proper formation of skin appendages; the functions of EDA2 and XEDAR are not known. EDA1 must be proteolytically processed to a soluble form to be active. Here, we show that treatment of pregnant Tabby mice with a recombinant form of EDA1, engineered to cross the placental barrier, permanently rescues the Tabby phenotype in the offspring. Notably, sweat glands can also be induced by EDA1 after birth. This is the first example of a developmental genetic defect that can be permanently corrected by short-term treatment with a recombinant protein.     

Non-syndromic tooth agenesis in two Chinese families associated with novel missense mutations in the TNF domain of EDA (ectodysplasin A)

Here we report two unrelated Chinese families with congenital missing teeth inherited in an X-linked manner. We mapped the affected locus to chromosome Xp11-Xq21 in one family. In the defined region, both families were found to have novel missense mutations in the ectodysplasin-A (EDA) gene. The mutation of c.947A>G caused the D316G substitution of the EDA protein. The mutation of c.1013C>T found in the other family resulted in the Thr to Met mutation at position 338 of EDA. The EDA gene has been reported responsible for X-linked hypohidrotic ectodermal dysplasia (XLHED) in humans characterized by impaired development of hair, eccrine sweat glands, and teeth. In contrast, all the affected individuals in the two families that we studied here had normal hair and skin. Structural analysis suggests that these two novel mutants may account for the milder phenotype by affecting the stability of EDA trimers. Our results indicate that these novel missense mutations in EDA are associated with the isolated tooth agenesis and provide preliminary explanation for the abnormal clinical phenotype at a molecular structural level.     

Molecular and therapeutic characterization of anti-ectodysplasin A receptor (EDAR) agonist monoclonal antibodies

The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC(50) of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments.     

Sequence polymorphisms of the EDA and the DL genes in the patients with an X-linked and an autosomal forms of anhidrotic ectodermal dysplasia

Oligodontia, sparse hair and deficiency of eccrine sweat glands are the features characteristic for the phenotype of the patients with anhidrotic ectodermal dysplasia (EDA). This syndrome is caused by mutations in the EDA or DL (downless) genes, encoding members of the TNF ligand and TNF receptor families, involved in the communication between the cells during embryonic life. We investigated both the coding and noncoding regions of the EDA and the DL genes in the patients exhibiting clinical symptoms of ectodermal dysplasia. Sequence analysis of the amplified fragments of the EDA gene revealed polymorphisms in introns three, four and five. The polymorphism in intron four was found in about 60% of the patients and was no more frequent than in the normal individuals. The two other polymorphisms were rare. Polymorphisms were also observed in exons 9 and 12 of the DL gene, but they did not alter the sequence of the protein product of the gene. Our results indicate that in order to accelerate screening for the mutations of the EDA gene and reduce the costs, the amplified fragments should not contain intronic sequences. However, in the case of the DL gene, where polymorphic sites are located in exons, restriction analysis with the use of appropriate enzyme should be conducted, but usually sequencing analysis could not be avoided.     

Definitive evidence for an autosomal recessive form of hypohidrotic ectodermal dysplasia clinically indistinguishable from the more common X-linked disorder.

A crucial issue in genetic counseling is the recognition of nonallelic genetic heterogeneity. Hypohidrotic (anhidrotic) ectodermal dysplasia (HED), a genetic disorder characterized by defective development of hair, teeth, and eccrine sweat glands, is usually inherited as an X-linked recessive trait mapped to the X-linked ectodermal dysplasia locus, EDA, at Xq12-q13.1. The existence of an autosomal recessive form of the disorder had been proposed but subsequently had been challenged by the hypothesis that the phenotype of severely affected daughters born to unaffected mothers in these rare families may be due to marked skewing of X inactivation. Five families with possible autosomal recessive HED have been identified, on the basis of the presence of severely affected females and unaffected parents in single sibships and in highly consanguineous families with multiple affected family members. The disorder was excluded from the EDA locus by the lack of its cosegregation with polymorphic markers flanking the EDA locus in three of five families. No mutations of the EDA gene were detected by SSCP analysis in the two families not excluded by haplotype analysis. The appearance of affected males and females in autosomal recessive HED was clinically indistinguishable from that seen in males with X-linked HED. The findings of equally affected males and females in single sibships, as well as the presence of consanguinity, support an autosomal recessive mode of inheritance. The fact that phenotypically identical types of HED can be caused by mutations at both X-linked and autosomal loci is analogous to the situation in the mouse, where indistinguishable phenotypes are produced by mutations at both X-linked (Tabby) and autosomal loci (crinkled and downless).     

Significant correction of disease after postnatal administration of recombinant ectodysplasin A in canine X-linked ectodermal dysplasia

Patients with defective ectodysplasin A (EDA) are affected by X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition characterized by sparse hair, inability to sweat, decreased lacrimation, frequent pulmonary infections, and missing and malformed teeth. The canine model of XLHED was used to study the developmental impact of EDA on secondary dentition, since dogs have an entirely brachyodont, diphyodont dentition similar to that in humans, as opposed to mice, which have only permanent teeth (monophyodont dentition), some of which are very different (aradicular hypsodont) than brachyodont human teeth. Also, clinical signs in humans and dogs with XLHED are virtually identical, whereas several are missing in the murine equivalent. In our model, the genetically missing EDA was compensated for by postnatal intravenous administration of soluble recombinant EDA. Untreated XLHED dogs have an incomplete set of conically shaped teeth similar to those seen in human patients with XLHED. After treatment with EDA, significant normalization of adult teeth was achieved in four of five XLHED dogs. Moreover, treatment restored normal lacrimation and resistance to eye and airway infections and improved sweating ability. These results not only provide proof of concept for a potential treatment of this orphan disease but also demonstrate an essential role of EDA in the development of secondary dentition.     

Generation and Characterization of Function-blocking Anti-ectodysplasin A (EDA) Monoclonal Antibodies That Induce Ectodermal Dysplasia

Development of ectodermal appendages, such as hair, teeth, sweat glands, sebaceous glands, and mammary glands, requires the action of the TNF family ligand ectodysplasin A (EDA). Mutations of the X-linked EDA gene cause reduction or absence of many ectodermal appendages and have been identified as a cause of ectodermal dysplasia in humans, mice, dogs, and cattle. We have generated blocking antibodies, raised in Eda-deficient mice, against the conserved, receptor-binding domain of EDA. These antibodies recognize epitopes overlapping the receptor-binding site and prevent EDA from binding and activating EDAR at close to stoichiometric ratios in in vitro binding and activity assays. The antibodies block EDA1 and EDA2 of both mammalian and avian origin and, in vivo, suppress the ability of recombinant Fc-EDA1 to rescue ectodermal dysplasia in Eda-deficient Tabby mice. Moreover, administration of EDA blocking antibodies to pregnant wild type mice induced in developing wild type fetuses a marked and permanent ectodermal dysplasia. These function-blocking anti-EDA antibodies with wide cross-species reactivity will enable study of the developmental and postdevelopmental roles of EDA in a variety of organisms and open the route to therapeutic intervention in conditions in which EDA may be implicated.     

High level production and one-step purification of biologically active ectodysplasin A1 and A2 immunoadhesins using the baculovirus/insect cell expression system

Ectodysplasin A (EDA) is a ligand of the tumor necrosis factor (TNF) family that has been shown to play a crucial role in ectodermal differentiation. Mutations of the syntenic ectodysplasin A gene (Eda) are responsible for Tabby (Ta) phenotype in mice and human X-linked hypohidrotic ectodermal dysplasia (XLHED). EDA-A1 and EDA-A2 are the two main splice variants of Eda, which differ from each other in only two amino acid residues and engage the tumor necrosis factor (TNF) family receptors EDAR and XEDAR, respectively. We have used the baculovirus/insect cell system to express the recombinant EDA proteins fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain. Immunoadhesins (4.5-4.7 mg/L) from crude supernatant could be purified to near homogeneity by using rProtein A affinity chromatography. The purified EDA immunoadhesins were endowed with ligand-binding activity as they could bind EDAR or XEDAR on the surface of 293T cells that had been transiently transfected with the corresponding plasmids. Functional activities of EDA immunoadhesins were demonstrated by their ability to activate the NF-kappaB pathway in cells expressing their cognate receptors. These results open up the possibility of obtaining large amounts of purified EDA proteins to investigate EDAR/XEDAR related signaling pathways and for the treatment of patients with X-linked hypohidrotic ectodermal dysplasia.     

Mutation identification in a canine model of X-linked ectodermal dysplasia

X-linked hypohidrotic ectodermal dysplasia (XHED), an inherited disease recognized in humans, mice, and cattle, is characterized by hypotrichosis, a reduced number or absence of sweat glands, and missing or malformed teeth. In a subset of affected individuals and animals, mutations in the EDA gene (formerly EDI), coding for ectodysplasin, have been found to cause this phenotype. Ectodysplasin is a homotrimeric transmembrane protein with an extracellular TNF-like domain, which has been shown to be involved in the morphogenesis of hair follicles and tooth buds during fetal development. Some human XHED patients also have concurrent immunodeficiency, due to mutations in the NF-κB essential modulator protein (IKBKG; formerly NEMO), which is also encoded on the X chromosome. In a breeding colony of dogs with XHED, immune system defects had been suspected because of frequent pulmonary infections and unexpected deaths resulting from pneumonia. To determine if defects in EDA or IKBKG cause XHED in the dogs, linkage analysis and sequencing experiments were performed. A polymorphic marker near the canine EDA gene showed significant linkage to XHED. The canine EDA gene was sequenced and a nucleotide substitution (G to A) in the splice acceptor site of intron 8 was detected in affected dogs. In the presence of the A residue, a cryptic acceptor site within exon 9 is used, leading to a frame shift and use of a premature stop codon that truncates the translation of both isoforms, EDA-A1 and EDA-A2, resulting in the absence of the TNF-like homology domain, the receptor-binding site of ectodysplasin.The sequence data described in this article have been submitted to GenBank under accession numbers AY924407–AY924414.     

Myodegeneration in EDA-A2 transgenic mice is prevented by XEDAR deficiency

EDA-A1 and EDA-A2 are members of the tumor necrosis factor family of ligands. The products of alternative splicing of the ectodysplasin (EDA) gene, EDA-A1 and EDA-A2 differ by an insertion of two amino acids and bind to distinct receptors. The longer isoform, EDA-A1, binds to EDAR and plays an important role in sweat gland, hair, and tooth development; mutations in EDA, EDAR, or the downstream adaptor EDARADD cause hypohidrotic ectodermal dysplasia. EDA-A2 engages the receptor XEDAR, but its role in the whole organism is less clear. We have generated XEDAR-deficient mice by gene targeting and transgenic mice expressing secreted forms of EDA-A1 or EDA-A2 downstream of the skeletal muscle-specific myosin light-chain 2 or skin-specific keratin 5 promoter. Mice lacking XEDAR were indistinguishable from their wild-type littermates, but EDA-A2 transgenic mice exhibited multifocal myodegeneration. This phenotype was not observed in the absence of XEDAR. Skeletal muscle in EDA-A1 transgenic mice was unaffected, but their sebaceous glands were hypertrophied and hyperplastic, consistent with a role for EDA-A1 in the development of these structures. These data indicate that XEDAR-transduced signals are dispensable for development of ectoderm-derived organs but might play a role in skeletal muscle homeostasis.     

Mutations in NPC1 Highlight a Conserved NPC1-Specific Cysteine-Rich Domain

Niemann-Pick type II disease is an autosomal recessive disorder characterized by a defect in intracellular trafficking of sterols. We have determined the intron/exon boundaries of eight exons from the conserved 3' portion of NPC1, the gene associated with most cases of the disease. SSCP analyses were designed for these exons and were used to identify the majority of mutations in 13 apparently unrelated families. Thirteen mutations were found, accounting for 19 of the 26 alleles. These mutations included eight different missense mutations (including one reported by Greer et al. [1998]), one 4-bp and two 2-bp deletions that generate premature stop codons, and two intronic mutations that are predicted to alter splicing. Two of the missense mutations were present in predicted transmembrane (TM) domains. Clustering of these and other reported NPC1 mutations in the carboxy-terminal third of the protein indicates that screening of these exons, by means of the SSCP analyses reported here, will detect most mutations. The carboxy-terminal half of the Npc1 protein shares amino acid similarity with the TM domains of the morphogen receptor Patched, with the largest stretch of unrelated sequence lying between two putative TM spans. Alignment of this portion of the human Npc1 protein sequence with Npc1-related sequences from mouse, yeast, nematode, and a plant, Arabidopsis, revealed conserved cysteine residues that may coordinate the structure of this domain. That 7 of a total of 13 NPC1 missense mutations are concentrated in this single Npc1-specific domain suggests that integrity of this region is particularly critical for normal functioning of the protein.     

Methylation State of the EDA Gene Promoter in Chinese X-Linked Hypohidrotic Ectodermal Dysplasia Carriers

Introduction

Hypodontia, hypohidrosis, sparse hair and characteristic faces are the main characters of X-linked hypohidrotic ectodermal dysplasia (XLHED) which is caused by genetic ectodysplasin A (EDA) deficiency. Heterozygous female carriers tend to have mild to moderate XLHED phenotype, even though 30% of them present no obvious symptom.

Methods

A large Chinese XLHED family was reported and the entire coding region and exon–intron boundaries of EDA gene were sequenced. To elucidate the mechanism for carriers’ tempered phenotype, we analyzed the methylation level on four sites of the promoter of EDA by the pyrosequencing system.

Results

A known frameshift mutation (c.573–574 insT) was found in this pedigree. Combined with the pedigrees we reported before, 120 samples comprised of 23 carrier females from 11 families and 97 healthy females were analyzed for the methylation state of EDA promoter. Within 95% confidence interval (CI), 18 (78.26%) carriers were hypermethylated at these 4 sites.

Conclusion

Chinese XLHED carriers often have a hypermethylated EDA promoter.