A new variant of the ectodysplasin A receptor death domain gene associated with anhidrotic ectodermal dysplasia in a Turkish family and its simple diagnosis by restriction fragment length polymorphism

ABSTRACT

Ectodermal dysplasia (ED), which exhibits a wide range of clinical symptoms, may be classified into three major types: hypohidrotic, anhidrotic, and hidrotic. A male child (proband) showing anhidrotic dysplasia was used as the subject of this study. The biopsy of the big toe revealed that the male child had no sweat glands. Genetic analysis of the patient revealed a mutation caused by a homozygous nucleotide substitution in the EDAR-associated death domain (EDARADD) (rs114632254) gene c.439G>A (p.Gly147Arg). Phenotypically, his teeth were sharp, but eight teeth were missing (oligodontia). The patient had normal nails with dry skin, sparse hair, everted lower lip vermilion, hyperpigmented eyelids, and abnormal nasal bridge morphology around the eyes. There is also a homozygous dominant (healthy) female and a heterozygous male in this family, who are cousins (aunt children) to the heterozygous parents. The daughter of the patient was also heterozygous. This mutation represents homozygous recessive inheritance, which we describe for the first time. Furthermore, we demonstrated that this genetic disorder can be readily diagnosed using the restriction fragment length polymorphism (RFLP) method after digestion with MnII restriction endonuclease.

INTRODUCTION

Ectodermal dysplasia (ED) patients display some developmental disorders, including disorders of the skin, hair, nails, teeth, and sweat glands, which develop from the ectoderm (Shoenfeld et al., 1975; Spfaer, 1981; Rodewald and Zahn-Messow, 1982; Keng, 1984; Potter and Bowie, 1984; Norval et al., 1988; Baskan et al., 2006). In afflicted individuals, hair may be weak and sparse, and teeth are abnormal and incomplete (Vierucci et al., 1994; Tape and Tye, 1995; Nijs and Huisman, 2001; Feng et al., 2007; Lexner et al., 2007; Baskan Ülkü and Yavuz, 2011; Callea et al., 2013). Baskan Ülkü and Yavuz, (2011) found that in 90% of ED patients, hair, eyebrows, and eyelashes were thin and sparse, whereas in 42%, the skin was soft, dry, flat, and thin because of a partial or complete lack of sweat and sebaceous glands. They also reported that ED patients may have defects, such as thickening, discoloration, dark pigmentation, and deformations in their nails.

The development of compromised skin and its derivatives may vary in some individuals (Freire-Maia et al., 1975). Hypohidrotic-type ED patients display slight sweaty conditions, whereas no sweating in anhidrotic type ED patients occurred because of the underdevelopment of sweat glands (Norval et al., 1988; Viljoen and Winship, 1988). In the absence of perspiration, children may have problems in controlling body temperature and a precarious high fever problem may occur (Nijs and Huisman, 2001; Segurado Rodríguez et al., 2002).

Over a hundred mutations associated with the ectodysplasin A (EDA), ectodysplasin A receptor (EDAR), and ectodysplasin A receptor death domain (EDARADD) genes have been described (Mikkola, 2009; Mousumi et al., 2013; Yin et al., 2013). In addition, WNT10A gene mutations also cause ED by inhibiting ectoderm development (Cluzeau et al., 2011; Martínez-Romero et al., 2019). Whereas missense, nonsense, and frameshift mutations are among the most detected mutations, deletion and splice points mutations are also encountered less frequently (Ersoy-Evans et al., 2006; Rifai et al., 2010; Farooq et al., 2013; Hayashi et al., 2013; Mousumi et al., 2013; Nikopensius et al., 2013; Schindler et al., 2013; Sun et al., 2013; Yin et al., 2013; Wang et al., 2014). A partial somatic mosaicism and translocation have been reported in some ED cases (Asamoah et al., 2003; Nishikomori et al., 2004; Kawai et al., 2012).

Ectodermal dysplasia is inherited in three different ways: X-linked recessive, autosomal recessive, and autosomal dominant (Mikkola, 2009; Baskan Ülkü and Yavuz, 2011). Although ED syndrome occurs by Mendelian inheritance, some studies have reported children born with ED from healthy parents as a result of a new mutation (Haghighi et al., 2013; Sun et al., 2013; Yang et al., 2013; Yoshioka et al., 2013). In many countries, however, prenatal diagnoses have been developed by identifying and detecting mutation types. Also, to prevent the disease, genetic counseling studies have been initiated (Shi et al., 2005; Li et al., 2006; Chen et al., 2012; Liu et al., 2013). An allele-specific oligonucleotide diagnostic method has been developed for prenatal diagnosis of ED-related diseases (Barbaro et al., 2012). Therefore, it is necessary to establish the mutational profile of a population to develop methods for accurate prenatal diagnosis (Chen et al., 2012).

The EDA gene has eight isoforms, localized on chromosome Xq13.1 (OMIM 300451) (http://omim.org), and translated to ectodysplasin A ligand protein (Monreal et al., 1998; Liu et al., 2012; Yin et al., 2013). EDAR is localized on chromosome 2q12.3 (OMIM 604095) and translated to the ectodysplasin A-receptor protein (Monreal et al., 1999; Mikkola, 2009). EDARADD (Gene locus MIM number 606603) is localized on chromosome 1q42–q43. The EDARADD gene has seven coding exons, exons 1a and 1b, which are alternatively spliced to yield isoforms of the ectodysplasin A receptor associated death domain A and ectodysplasin A receptor associated death domain B (Bal et al., 2007). Isoform A (OMIM 614940) and B (OMIM 614941) of the ECTD11B gene produced a receptor-associated protein containing 215 and 205 amino acids, respectively (https://www.ncbi.nlm.nih.gov/nuccore/1036031426). Comparing the two isoforms, there is only a difference between the amino acids originating from the first exon.

ED is a hereditary disease that yields many complex and different phenotypic symptoms. Detecting the ED syndrome and facilitating diagnosis are extremely important. Therefore, we examined the mutations in an anhidrotic ED patient and his family.

RESULTS

Based on a genetic analysis, a nucleotide exchange was identified in the c.439G>A region of the EDARADD gene (synonym: ECTD11A; ECTD11B; ED3; EDA3). The amplified and sequenced nucleotide sequences were compared using the NCBI database. The results showed the presence of a substitution mutation in the EDARADD gene at the last exon. This change was thought to be the cause of the disease through an in silico analysis and the patient was homozygous for this gene locus. This missense mutation changed the amino acid sequence of the synthesized protein at 147^th codon (p.Gly147Arg) (Table 1). According to the in-silico analysis, this change was found to be compatible with the disease phenotype.

Table 1. Nucleotide sequences of the exons of EDARADD gene (ECTD11B) and the amino acids of ectodysplasin A receptor associated death domain A

Nucleotide Sequence (648 bp):

Exon 1 (1-61, totally 61 bp)

ATGGGCCTCAGGACGACTAAACAGATGGGGAGAGGCACTAAAGCTCCTGGTCACCAAGAGG

Exon 2 (62-120, totally 59 bp)

ATCATATGGTAAAGGAACCAGTGGAAGACACAGACCCTAGCACTTTATCCTTTAATATG

Exon 3 (121-160, totally 40 bp)

TCAGACAAATATCCCATTCAAGATACGGAACTCCCTAAAG

Exon 4 (161-219, totally 59 bp)

CTGAAGAATGTGATACAATTACTTTGAACTGCCCACGAAATTCAGATATGAAAAATCAG

Exon 5 (220-265, totally 46 bp)

GGAGAAGAAAATGGCTTTCCAGATAGCACTGGAGATCCTCTTCCAG

Exon 6 (266-645, totally 380 bp and stop codon)

AGATCAGCAAGGACAACTCCTGCAAAGAAAACTGTACTTGTTCCTCCTGCTTGCTCCGGGCCCCCACCATAAGTGACTTGCTCAATGATCAGGACTTACTAGACGTGATCAGGATAAAGCTGGATCCGTGTCACCCAACGGTGAAAAACTGGAGGAATTTTGCAAGCAAATGGG(A)*GGATGTCCTATGACGAATTGTGCTTCCTGGAGCAGAGGCCACAGAGCCCCACCTTGGAGTTCTTGCTCCGGAACAGTCAGAGGACGGTGGGCCAGCTGATGGAGCTCTGCAGGCTCTACCACAGGGCCGACGTGGAGAAGGTTCTGCGCAGGTGGGTGGACGAGGAGTGGCCCAAGCGGGAGCGTGGAGACCCCTCCAGGCACTTCTAG

Translation (215 aa):

MGLRTTKQIGRGTKAPGHQEDHMVKEPVEDTDPSTLSFNMSDKYPIQDTELPKAEECDTITLNCPRNSDMKNQGEENGFPDSTGDPLPEISKDNSCKENCTCSSCLLRAPTISDLLNDQDLLDVIRIKLDPCHPTVKNWRNFASKWG(R)*MSYDELCFLEQRPQSPTLEFLLRNSQRTVGQLMELCRLYHRADVEKVLRRWVDEEWPKRERGDPSRHF(https://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi?REQUEST=CCDS&GO=MainBrowse&DATA=CCDS1610.1)

*  Parenthetical: Substituted nucleotide and new amino acid.

In a mutational screen of the family members, mutations showed Mendelian inheritance and were inherited in a recessive manner (Fig. 1). The parents and the youngest child (I-1, I-2 and II-4) were carriers for this mutation, whereas the daughter was homozygous normal (II-3). The patient’s wife was also homozygous dominant (healthy), whereas the patient’s daughter was a carrier as expected (II-1 and III-1). The Sanger DNA sequencing confirmed the homozygous normal individual, the patient, and the heterozygous individuals carrying this mutation (Fig. 2).

Fig. 1. Family pedigree for autosomal recessive inheritance of ectodermal dysplasia.

Fig. 2. Sequencing results of homozygous normal (A), patient (B), and heterozygotes (C).

To identify the mutation point, the amplification product (128 bp) (Fig. 3A) was cut with the MnII restriction enzyme to obtain 72 and 56 bp fragments in the healthy donors, because there is another cutoff region outside the expected mutation within this amplification region. The MnII restriction endonuclease recognizes the 5’-GAGG-3’ sequence with an altered A base at the mutation point (5’-(N)₆GAGG-3’/3’-(N)₇CTCC-5’) and cuts the DNA strand before nucleotides #6 (5’) and #7 (3’). If the gene was normal (GGGG), MnII-cutting would not be possible (http://nc2.neb.com/NEBcutter2/).

Fig. 3. Amplification products (A) and band profiles after MnII restriction endonuclease digestion (B), (M: Marker 100 bp, 1: Heterozygous, 2: Homozygous normal, and 3: Patient).

After MnII digestion, the two expected fragments of 72 and 56 bp were obtained in healthy individuals. In carriers, a total of four fragments consisting of 72, 56, 35, and 21 bp were evident. In the patient, a 56 bp fragment was not observed as only 72, 35, and 21 bp fragments were detected (Fig. 3B).

The digestion profiles obtained with the MnII restriction endonuclease confirm the results shown in Fig. 2. Individual II-2 is an anhidrotic ED patient and individuals II-1 and II-3 are homozygous normal. The others are carriers for this mutation.

DISCUSSION

EDARADD encodes two isoforms, A and B (ectodysplasin A receptor associated death domain A and B), which are formed by alternative splicing of the first exon. Only the N-terminal end of the protein synthesized by these two isoforms is different (Yan et al., 2002). The EDAR gene encodes a receptor protein present on the cell surface for the ectodysplasin A ligand, which is synthesized by the EDA gene. This signal is transmitted to the EDARADD protein, which functions as an adapter to activate the nuclear factor kappa-B (NF-kB) signaling pathway to initiate the dermal development (Bibi et al., 2011).

The mutation detected for the EDARADD gene c.439G>A (p.Gly147Arg) is inherited recessively based on Mendelian rules and prevents the development of sweat glands in patients. Isoform A of the EDARADD gene has six coding exons (totally 648 nucleotides in length) encoding a receptor-associated protein that contains 215 amino acids (Table 1) (https://www.ncbi.nlm.nih.gov/nuccore/1036031426). The mutated nucleotide is localized to position 236482440 of chromosome 1 (https://www.ncbi.nlm.nih.gov/gene/128178).

We identified a substitution in the sixth exon of the ectodermal dysplasia EDARADD gene. Guanine was converted to an adenine at the 439^th nucleotide (c.439G>A). This variant changed the 147^th codon (GGG) in the mRNA to a new codon (AGG), which converted a Gly amino acid residue to an Arg residue (p.Gly147Arg) (Table 1, Parenthetical). Based on HGVS-nomenclature [Human Genom Variation Society (http://varnomen.hgvs.org)], the variant was NM_145861.4:c.439G>A,p.(Gly147Arg). This new mutant protein prevents the transmission of intracellular signals to the NF-kB protein.

Nuclear factor kappa-B (NF-kB) is an essential signaling protein for skin development. EDARADD is a downstream stimulator of tumor necrosis factor receptor (TNFR)-related genes. EDARADD signals transmitted to the cytoplasm are processed by the signal transducers TRAF6, TAB2, and TAK1 and transmitted to the NF-kB protein, which is a transcription factor located within the nucleus. NF-kB regulates target gene expression (Morlon et al., 2005; Mikkola, 2009; Lefebvre and Mikkola, 2014). Based on our results, the newly discovered mutation inhibits the function of EDARADD and prevents the transmission of intracellular signals to NF-kB. Therefore, transcription of target genes responsible for the development of sweat glands is not possible, because Gly is a nonpolar hydrophobic amino acid, whereas Arg is positively charged and hydrophilic. This hydrophobic-hydrophilic exchange disrupts the globular structure of the protein synthesized by the EDARADD gene and cannot transmit a signal through the EDAR receptor (Aydin et al., 2017).

In the present study, a gene mutation, a possible protein change, and familial inheritance patterns of the mutation were examined. Additionally, the missense change occurring the amino acids are thought to be the cause of the nonfunctional protein structure. However, because of the new mutation, the data are not yet included in the population database. The CADD score was 31, which indicated that the variant was in the top 1% of the most deleterious mutations in the human genome as described previously (Chr 1, Position 236482440, CADD GRCh38-v1.5) (Rentzsch et al., 2019; Ahmed et al., 2021). This variant was classified as likely pathogenic based on the guidelines of the American College of Medical Genetics and Genomics (ACMG) (Richards et al., 2015). We established a molecular assay that a healthcare provider can use in decision-making.

A total of 17 variants were detected in the EDARADD gene, of which most are pathogenic and a cause of disease (Table 2). In addition, 400 variants of the EDARADD gene have been identified in the gnomAD database (https://gnomad.broadinstitute.org/gene/ENSG00000186197?dataset=gnomad_r2_1).

Table 2. Variants of the EDARADD gene associated with ectodermal dysplasia

Location	Accession no	SNP ID	Significance	Resulting a.a. change	References
c.27G>A*		rs966365	Benign	p.Met9Ile	Wohlfart et al., 2016b
c.60G>A	NM_145861.2	rs60808129	Benign	p.Glu20=	https://www.malacards.org
c.120G>A	NM_145861.2	rs879255553	Pathogenic	Second intron, affect 5’splice	Chaudhary et al., 2016
c.157insA	NM145861.2		Pathogenic	p.Ala54Serfs*2	Koguchi-Yoshioka et al., 2015
c.278C>T	NM_080738.3	rs114632254	pathogenic	p.Ser103 Phe	Salvi et al., 2016
c.308C>T	NM_145861.2	rs114632254	Benign	p.Ser103Phe	Martínez-Romero et al., 2019
c.328G>T	NM_080738.3		Pathogenic	p.Asp110Tyr	Cluzeau et al., 2011
c.335T>G*	NM_080738		Pathogenic	p.Leu112Arg	Bal et al., 2007
c.361C>T	NM_080738		Pathogenic	p.Pro121Ser	Suda et al., 2010
c.367G>A	NM_145861.2		Pathogenic	p.Asp123Asn	Wohlfart et al., 2016a
c.369C>T		rs604070	Benign	No change	Wohlfart et al., 2016b
c.393G>A	NM_145861.2	rs139996586	Benign	p.Pro131=	https://www.malacards.org
c.402-407del	NM_145861		Pathogenic	p.Thr135-Val136del	Chassaing et al., 2010
c.417G>A	NM_145861.2	rs954823206	Pathogenic	p.Trp139Ter	https://www.malacards.org
c.424G>A	NM_080738.3	rs74315309	Pathogenic	p. Glu142Lys	Headon et al., 2001
c.508C>T			Pathogenic	p.Arg170Trp	Chaudhary et al., 2016
c.509G>A	NM_145861.2	rs757261515	Uncertain	p.Arg170Gln	https://www.malacards.org

*  Mutations in ECTD11A.

Thirty-nine variants were detected in the 5’-UTR and 3’-UTR regions, and 112 variants were identified as missense. Of these, four were contained a loss of the start codon. A total of 187 variants were found within the intron regions, whereas 24 occurred in splice regions. The missense variant reported in the current study is novel and there is no other data that appears in the previous studies and databases, such as PubMed/Medline, OMIM, gnomAD, ClinVar, or other databases. As shown, the c.439G>A mutation was identified for the first time for this gene. Furthermore, it was readily identified by RFLP using the MnII restriction endonuclease without sequencing. As a result, the gene mutation that was detected is inherited recessively and anhidrotic ED syndrome is caused by completely preventing the formation of sweat glands in the homozygous form.

CONCLUSION

We detected a new mutation in the EDARADD gene that is associated with recessive inheritance Ectodermal dysplasia. Genetic studies conducted in both the patient and family revealed that the mutation was inherited recessively according to Mendelian rules. This variant was classified as likely pathogenic according to ACMG guidelines.

MATERIALS AND METHODS

This study was conducted in agreement with the statement on the Declaration of Helsinki and conducted with ethical approval from the Ethics Committee of Non-Interventional Research (approval date and number: 27.06.2019/11-14) of Firat University, Elazig, Turkey.

Phenotypic abnormalities of the proband were named according to the human phenotype ontology identifier criteria (https://hpo.jax.org/app/).

The oldest child in the family was diagnosed with anhidrotic ED (Fig. 1, II-2). The proband birth weight was 2700 grams. He consulted a doctor several times for high fever. Additionally, some drugs were prescribed for symptomatic treatment. The proband did not sweat when he was a year old and tooth eruption began at 8 months. The proband had no neonatal/childhood history, such as recurrent respiratory tract infections, atopic eczema, gastroesophageal reflux, recurrent pneumonia, or dry-eye-related symptoms. Currently, the patient is 28 years old with sparse hair, dry skin, everted lower lip vermilion, hyperpigmentation around the eyes, and an abnormal nasal bridge morphology. Upon examination, his teeth were sharp and eight of them were missing (oligodontia); however, his nails were normal (without dysplastic nails). He exhibited an anhidrotic phenotype because there were no sweat glands according to a pathological analysis.

EDTA-containing blood samples were collected from family members who had a male anhidrotic ED patient (proband) (Fig. 1, II-2). This family had a total of three children and a grandchild (Fig. 1, II-2-4). The wife of the patient (Fig. 1, II-1) was also included in this study. Genomic DNA was isolated from the blood samples of volunteers using the Axygen DNA isolation kit (Thermo Scientific, GeneJet Genomic DNA Purification Kit, Lithuania) as described by the manufacturer. The genomic DNA was amplified using a Verity 96-well thermal cycler (Applied Biosystems, USA). Genomic DNA specimens were stored at −20 ℃ until analysis.

A genetic analysis was performed over a wide area because many genes are associated with anhidrotic ED (Intergene Genetic Center). Because many genes affect ED disease, SNPs were first screened using an in silico computer simulation at Intergen laboratories to facilitate the experimental studies. Missense SNPs in the EDA, EDAR, and EDARADD genes were detected by scanning the NCBI dbSNP database. PolyPhen-2 (Polymorphism Phenotyping v2) internet-based software tools were used to identify the genes with missense mutations from these SNPs. Subsequently, all SNPs that could theoretically cause missense mutations were examined in both of the patients and parents through experimental studies. First, only the patient’s DNA was amplified and sequenced. In total, 4800 bp were sequenced for all exons and the UTR regions of the three ED genes (EDA, EDAR, EDARADD) using Next Generation Sequencing Method (Illumina-MISEQ-San Diego). The other family members and the patient’s wife were screened for the respective mutation and the family’s inheritance pattern was determined.

Experimental studies carried out on the detected SNP. To confirm the detected mutations, three amplicons (patient, heterozygous, and homozygous) were sequenced using the Sanger DNA sequencing protocol. The allele frequency in the general population of the variant was confirmed in the Genome Aggregation Database (gnomAD) (Karczewski et al., 2020). Pathogenicity was classified according to the ACMG/American Molecular Pathology (Richards et al., 2015) guidelines. The novel variant was registered in the ClinVar database as accession number SCV002817373 (Landrum et al., 2018).

To detect mutations by the RFLP method, a total of 128 base pair amplifications were performed in the 6^th exon using the following primers: Forward 5’-tgacttgctcaatgatcaggac-3’ and Reverse 5’-ggaagcacaattcgtcatagga-3’. A 25 μL PCR mixture was prepared that included approximately 125 ng gDNA, 2.5 μL of each dNTP, 0.5 μl (50 pmol) of both primers, 2.5 μL 10× Taq buffer, 2 μL MgCI₂ (25 mM), and 1.5 μL Taq polymerase (Sigma-Aldrich, St. Louis, MO, USA). The following PCR cycling conditions were used: an initial denaturation step of 5 min at 95 ℃; 30 cycles of 45 s at 95 ℃, 45 s at 59 ℃ for annealing, 45 s at 72 ℃ for elongation; and a final elongation step of 7 min at 72 ℃. After amplification, the PCR products (amplicons) were subject to electrophoresis (1.5% agarose gel) and the restriction enzyme MnII (Thermo Fisher Scientific, ER1071) was used to distinguish the c.439G>A (p.Gly147Arg) genotype. This easily determined the presence of the mutation without sequencing.

MnII restriction digestion was done according to the manufacturer’s instructions (https://www.thermofisher.com/order/catalog/product/ER1071). For this purpose, 15 μL of PCR products were mixed with 2 μL MnII (300 unit), 2 μL 10× buffer, and 11 μL distilled sterile water. The mixture was incubated at 37 ℃ for 16 h followed by electrophoresis on a 4% agarose gel and detection.

DECLARATIONS

Funding: This study was not supported by any institution.

Ethical Considerations: This study was conducted in agreement with the statement on the Declaration of Helsinki and received ethical approval. All participants and their legal guardians were informed beforehand and informed consent was obtained.

Data Availability Statement: The reports and data for this study may be requested from the author; however, the genetic material and information of the subjects cannot be provided. The data are not publicly available because of privacy or ethical restrictions.

ACKNOWLEDGMENTS

The author thanks to Prof. Dr. Suleyman Bayram for scientific collaborations and to Ass. Prof. Dr. Askin Sen and Assoc. Prof. Dr. Serdar Ceylaner for in silico analysis.

REFERENCES

•  Ahmed,  H. A.,  El-Kamah,  G. Y.,  Rabie,  E.,  Mostafa,  M. I.,  Abouzaid,  M. R.,  Hassib,  N. F.,  Mehrez,  M. I.,  Abdel-Kader,  M. A.,  Mohsen,  Y. H.,  Zada,  S. K., et al. (2021) 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. Genes (Basel) 12, 1389.
•  Asamoah,  A.,  Decker,  A. B.,  Wiktor,  A., and  Van Dyke,  D. L. (2003) Child with De Novo t(1;6)(p22.1;p22.1) translocation and features of ectodermal dysplasia with hypodontia and developmental delay. Am. J. Med. Genet. A 118A, 82–85.
•  Aydin,  M.,  Rencuzogullari,  E.,  Bayram,  S.,  Sevgiler,  Y., and  Genc,  A. (2017) Alterations on high HbF levels may be associated with KLF1 gene mutations. Cell. Mol. Biol. 63, 51–57.
•  Bal,  E.,  Baala,  L.,  Cluzeau,  C.,  El Kerch,  F.,  Ouldim,  K.,  Hadj-Rabia,  S.,  Bodemer,  C.,  Munnich,  A.,  Courtois,  G.,  Sefiani,  A., et al. (2007) Autosomal dominant anhidrotic ectodermal dysplasias at the EDARADD locus. Hum. Mutat. 28, 703–709.
•  Barbaro,  V.,  Confalonieri,  L.,  Vallini,  I.,  Ferrari,  S.,  Ponzin,  D.,  Mantero,  G.,  Willoughby,  C. E.,  Parekh,  M., and  Di Iorio,  E. (2012) Development of an allele-specific real-time PCR assay for discrimination and quantification of p63 R279H mutation in EEC syndrome. J. Mol. Diagn. 14, 38–45.
•  Baskan Ülkü,  S. Z., and  Yavuz,  I. (2011) Ektodermal displazili hastalarda ptotetik yaklasimlar (Prosthetic approaches in ectodermal dysplasia patients). J. Dent. Fac. Atatürk Uni. 21, 57–61 (in Turkish).
•  Baskan,  Z.,  Yavuz,  I.,  Ulku,  R.,  Kaya,  S.,  Yavuz,  Y.,  Basaran,  G.,  Adiguzel,  O., and  Ozer,  T. (2006) Evaluation of ectodermal dysplasia. Kaohsiung J. Med. Sci. 22, 171–176.
•  Bibi,  N.,  Ahmad,  S.,  Ahmad,  W., and  Naeem,  M. (2011) Molecular genetic analysis of consanguineous Pakistani families with autosomal recessive hypohidrotic ectodermal dysplasia. Australas J. Dermatol. 52, 37–42.
•  Callea,  M.,  Teggi,  R.,  Yavuz,  I.,  Tadini,  G.,  Priolo,  M.,  Crovella,  S.,  Clarich,  G., and  Grasso,  D. L. (2013) Ear nose throat manifestations in hypoidrotic ectodermal dysplasia. Int. J. Pediatr. Otorhinolaryngol. 77, 1801–1804.
•  Chassaing,  N.,  Cluzeau,  C.,  Bal,  E.,  Guigue,  P.,  Vincent,  M.-C.,  Viot,  G.,  Ginisty,  D.,  Munnich,  A.,  Smahi,  A., and  Calvas,  P. (2010) Mutations in EDARADD account for a small proportion of hypohidrotic ectodermal dysplasia cases. Br. J. Dermatol. 162, 1044–1048.
•  Chaudhary,  A. K.,  Girisha,  K. M., and  Bashyam,  M. D. (2016) A novel EDARADD 5’-splice site mutation resulting in activation of two alternate cryptic 5’-splice sites causes autosomal recessive Hypohidrotic Ectodermal Dysplasia. Am. J. Med. Genet. A 170, 1639–1641.
•  Chen,  L.,  Zhao,  Y.-y.,  Wei,  Y.,  Wang,  Y.,  Zhang,  Y.,  Wang,  Y.-q.,  Liu,  J.-y.,  Yang,  Y., and  Tan,  Y.-h. (2012) Prenatal diagnosis of anhidrotic ectodermal dysplasia with unconventional loci abnormalities, a case report. Chin. Med. J. 125, 3177–3179.
•  Cluzeau,  C.,  Hadj-Rabia,  S.,  Jambou,  M.,  Mansour,  S.,  Guigue,  P.,  Masmoudi,  S.,  Bal,  E.,  Chassaing,  N.,  Vincent,  M.-C.,  Viot,  G., et al. (2011) Only four genes (EDA1, EDAR, EDARADD, and WNT10A) account for 90% of hypohidrotic/anhidrotic ectodermal dysplasia cases. Hum. Mutat. 32, 70–72.
•  Ersoy-Evans,  S.,  Erkin,  G.,  Fassihi,  H.,  Chan,  I.,  Paller,  A. S.,  Sürücü,  S., and  McGrath,  J. A. (2006) Ectodermal dysplasia-skin fragility syndrome resulting from a new homozygous mutation, 888delC, in the desmosomal protein plakophilin 1. J. Am. Acad. Dermatol. 55, 157–161.
•  Farooq,  M.,  Kurban,  M.,  Fujimoto,  A.,  Fujikawa,  H.,  Abbas,  O.,  Nemer,  G.,  Saliba,  J.,  Sleiman,  R.,  Tofaili,  M.,  Kibbi,  A.-G., et al. (2013) A homozygous frameshift mutation in the HOXC13 gene underlies pure hair and nail ectodermal dysplasia in a Syrian family. Hum. Mutat. 34, 578–581.
•  Feng,  H.-l.,  Zhang,  X.-x., and  Wu,  H. (2007) Research advances in tooth agenesis. Journal of Peking University, Health sciences 39, 13–17 (in Chinese).
•  Freire-Maia,  N.,  Fortes,  V. A.,  Pereira,  L. C.,  Opitz,  J. M.,  Marcalle,  F. A., and  Cavalli,  I. J. (1975) A syndrome of hypohidrotic ectodermal dysplasia with normal teeth, peculiar facies, pigmentary disturbances, psychomotor and growth retardation, bilateral nuclear cataract, and other signs. J. Med. Genet. 12, 308–310.
•  Haghighi,  A.,  Nikuei,  P.,  Haghighi-Kakhki,  H.,  Saleh-Gohari,  N.,  Baghestani,  S.,  Krawitz,  P. M.,  Hecht,  J., and  Mundlos,  S. (2013) Whole-exome sequencing identifies a novel missense mutation in EDAR causing autosomal recessive hypohidrotic ectodermal dysplasia with bilateral amastia and palmoplantar hyperkeratosis. Br. J. Dermatol. 168, 1353–1356.
•  Hayashi,  R.,  Farooq,  M.,  Fujikawa,  H.,  Fujimoto,  A.,  Hashimoto,  T.,  Ito,  M., and  Shimomura,  Y. (2013) Case of hypohidrotic ectodermal dysplasia caused by a large deletion mutation in the EDA gene. J. Dermatol. 40, 281–283.
•  Headon,  D. J.,  Emmal,  S. A.,  Ferguson,  B. M.,  Tucker,  A. S.,  Justice,  M. J.,  Sharpe,  P. T.,  Zonana,  J., and  Overbeek,  P. A. (2001) Gene defect in ectodermal dysplasia implicates a death domain adapter in development. Nature 414, 913–916.
•  Karczewski,  K. J.,  Francioli,  L. C.,  Tiao,  G.,  Cummings,  B. B.,  Alföldi,  J.,  Wang,  Q.,  Collins,  R. L.,  Laricchia,  K. M.,  Ganna,  A.,  Birnbaum,  D. P., et al. (2020) The mutational constraint spectrum quantified from variation in 141,456 humans. Nature 581, 434–443.
•  Kawai,  T.,  Nishikomori,  R.,  Izawa,  K.,  Murata,  Y.,  Tanaka,  N.,  Sakai,  H.,  Saito,  M.,  Yasumi,  T.,  Takaoka,  Y.,  Nakahata,  T., et al. (2012) Frequent somatic mosaicism of NEMO in T cells of patients with X-linked anhidrotic ectodermal dysplasia with immunodeficiency. Blood 119, 5458–5466.
•  Keng,  S. B. (1984) Oro-facial manifestation of hypohidrotic ectodermal dysplasia—case report. Ann. Acad. Med. Singap. 13, 552–555.
•  Koguchi-Yoshioka,  H.,  Wataya-Kaneda,  M.,  Yutani,  M.,  Nakano,  H.,  Sawamura,  D., and  Katayama,  I. (2015) Partial anhidrosis demonstrated by Q-SART in a patient with a novel mutation in the EDARADD gene. J. Eur. Acad. Dermatol. Venereol. 29, 1443–1444.
•  Landrum,  M. J.,  Lee,  J. M.,  Benson,  M.,  Brown,  G. R.,  Chao,  C.,  Chitipiralla,  S.,  Gu,  B.,  Hart,  J.,  Hoffman,  D.,  Jang,  W., et al. (2018) ClinVar: improving access to variant interpretations and supporting evidence. Nucleic Acids Res. 46, D1062–D1067.
•  Lefebvre,  S., and  Mikkola,  M. L. (2014) Ectodysplasin research—where to next? Semin. Immunol. 26, 220–228.
•  Lexner,  M. O.,  Bardow,  A.,  Hertz,  J. M.,  Nielsen,  L. A., and  Kreiborg,  S. (2007) Anomalies of tooth formation in hypohidrotic ectodermal dysplasia. Int. J. Paediatr. Dent. 17, 10–18.
•  Li,  W.,  Gao,  B.-d.,  Li,  L.-y.,  Xiao,  H. M., and  Lu,  G.-x. (2006) Mutation screening and prenatal diagnosis of hidrotic ectodermal dysplasia in a Chinese family. Chinese journal of medical genetics 23, 618–621 (in Chinese).
•  Liu,  N.,  Shi,  H.-r.,  Wu,  Q.-h.,  Jiang,  M., and  Kong,  X.-d. (2013) Mutation analysis and first-trimester prenatal diagnosis for a Chinese family with hidrotic ectodermal dysplasia. Chinese journal of medical genetics 30, 407–409.
•  Liu,  Y.,  Yu,  X.,  Wang,  L.,  Li,  C.,  Archacki,  S.,  Huang,  C.,  Liu,  J. Y.,  Wang,  Q.,  Liu,  M., and  Tang,  Z. (2012) Mutation p.Leu354Pro in EDA causes severe hypohidrotic ectodermal dysplasia in a Chinese family. Gene 491, 246–250.
•  Martínez-Romero,  M. C.,  Ballesta-Martínez,  M. J.,  López-González,  V.,  Sánchez-Soler,  M. J.,  Serrano-Antón,  A. T.,  Barreda-Sánchez,  M.,  Rodriguez-Peña,  L.,  Martínez-Menchon,  M. T.,  Frías-Iniesta,  J.,  Sánchez-Pedreño,  P., et al. (2019) EDA, EDAR, EDARADD and WNT10A allelic variants in patients with ectodermal derivative impairment in the Spanish population. Orphanet J. Rare Dis. 14, 281–291.
•  Mikkola,  M. L. (2009) Molecular aspects of hypohidrotic ectodermal dysplasia. Am. J. Med. Genet. A 149A, 2031–2036.
•  Monreal,  A. W.,  Ferguson,  B. M.,  Headon,  D. J.,  Street,  S. L.,  Overbeek,  P. A., and  Zonana,  J. (1999) Mutations in the human homologue of mouse dl cause autosomal recessive and dominant hypohidrotic ectodermal dysplasia. Nat. Genet. 22, 366–369.
•  Monreal,  A. W.,  Zonana,  J., and  Ferguson,  B. (1998) Identification of a new splice form of the EDA1 gene permits detection of nearly all X-linked hypohidrotic ectodermal dysplasia mutations. Am. J. Hum. Genet. 63, 380–399.
•  Morlon,  A.,  Munnich,  A., and  Smahi,  A. (2005) TAB2, TRAF6 and TAK1 are involved in NF-κB activation induced by the TNF-receptor, Edar and its adaptator Edaradd. Hum. Mol. Genet. 14, 3751–3757.
•  Mousumi,  T.,  Xiong,  Z.,  Lu,  L.,  Liu,  S.,  Xia,  K., and  Hu,  Z. (2013) Identification of a known GJB6 mutation in an autosomal dominant inherited Chinese family with hidrotic ectodermal dysplasia. Journal of Central South University, Medical sciences 38, 761–765.
•  Nijs,  E. L., and  Huisman,  T. A. (2001) Recurrent fever and lack of tooth buds. A case of ectodermal dysplasia in a 9 months old boy. JBR-BTR 84, 256–257.
•  Nikopensius,  T.,  Annilo,  T.,  Jagomägi,  T.,  Gilissen,  C.,  Kals,  M.,  Krjutškov,  K.,  Mägi,  R.,  Eelmets,  M.,  Gerst-Talas,  U.,  Remm,  M., et al. (2013) Non-syndromic tooth agenesis associated with a nonsense mutation in ectodysplasin-A (EDA). J. Dent. Res. 92, 507–511.
•  Nishikomori,  R.,  Akutagawa, H.,  Maruyama,  K.,  Nakata-Hizume,  M.,  Ohmori,  K.,  Mizuno,  K.,  Yachie,  A.,  Yasumi,  T.,  Kusunoki,  T.,  Heike,  T., et al. (2004) X-linked ectodermal dysplasia and immunodeficiency caused by reversion mosaicism of NEMO reveals a critical role for NEMO in human T-cell development and/or survival. Blood 103, 4565–4572.
•  Norval,  E. J.,  van Wyk,  C. W.,  Basson,  N. J., and  Coldrey,  J. (1988) Hypohidrotic ectodermal dysplasia: a genealogic, stereomicroscope, and scanning electron microscope study. Pediatr. Dermatol. 5, 159–166.
•  Potter,  P. C., and  Bowie,  M. D. (1984) Dysphagia in hypohidrotic ectodermal dysplasia. A case report. S. Afr. Med. J. 66, 232–234.
•  Rentzsch,  P.,  Witten,  D.,  Cooper,  G. M.,  Shendure,  J., and  Kircher,  M. (2019) CADD: predicting the deleteriousness of variants throughout the human genome. Nucleic Acids Res. 47, D886–D894.
•  Richards,  S.,  Aziz,  N.,  Bale,  S.,  Bick,  D.,  Das,  S.,  Gastier-Foster,  J.,  Grody,  W. W.,  Hegde,  M.,  Lyon,  E.,  Spector,  E., et al. (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 17, 405–424.
•  Rifai,  L.,  Port-Lis,  M.,  Tabet,  A. C.,  Bailleul-Forestier,  I.,  Benzacken,  B.,  Drunat,  S.,  Kuzbari,  S.,  Passemard,  S.,  Verloes,  A., and  Aboura,  A. (2010) Ectodermal dysplasia-like syndrome with mental retardation due to contiguous gene deletion: further clinical and molecular delineation of del(2q32) syndrome. Am. J. Med. Genet. A 152A, 111–117.
•  Rodewald,  A., and  Zahn-Messow,  K. (1982) Dermatoglyphics findings in families with X-linked hypohidrotic (or anhidrotic) ectodermal dysplasia (HED). Prog. Clin. Biol. Res. 84, 451–458.
•  Salvi,  A.,  Giacopuzzi,  E.,  Bardellini,  E.,  Amadori,  F.,  Ferrari,  L.,  De Petro,  G.,  Borsani,  G., and  Majorana,  A. (2016) Mutation analysis by direct and whole exome sequencing in familial and sporadic tooth agenesis. Int. J. Mol. Med. 38, 1338–1348.
•  Schindler,  A.,  Guazzarotti,  L.,  Mameli,  C.,  Urbani,  E.,  Mozzanica,  F.,  Guerrini,  L., and  Zuccotti,  G. V. (2013) Vomer aplasia in a patient carrying a de novo mutation of the TP63 gene (3q27). Int. J. Pediatr. Otorhinolaryngol. 77, 1606–1608.
•  Segurado Rodríguez,  M. A.,  Ortiz De Frutos,  F. J,  Cornejo Navarro,  P.,  Rodríguez Peralto,  J. L.,  Sánchez Del Pozo,  J.,  Guerra Tapia,  A., and  Iglesias Díez,  L. (2002) Hypohidrotic ectodermal dysplasia: a cause of fever of unknown origin. An. Esp. Pediatr. 56, 253–257 (in Spanish).
•  Shi,  H.-j.,  Fang,  Q., and  Wang,  L.-t. (2005) Prenatal diagnosis of X-linked anhidrotic ectodermal dysplasia with X-chromosome inversion. Chin Med. J. 85, 1845–1848 (in Chinese).
•  Shoenfeld,  Y.,  Shapiro,  Y.,  Fisher,  B. K., and  Dvoretzky,  I. (1975) Sweating in the ‘anhidrotic type’ of congenital ectodermal dysplasia. Dermatologica 150, 277–282.
•  Spfaer,  J. A. (1981) A dental approach to carrier screening in X linked hypohidrotic ectodermal dysplasia. J. Med. Genet. 18, 459–460.
•  Suda,  N.,  Bazar,  A.,  Bold,  O.,  Jigjid,  B.,  Garidkhuu,  A.,  Ganburged,  G., and  Moriyama,  K. (2010) A Mongolian patient with hypohidrotic ectodermal dysplasia with a novel P121S variant in EDARADD. Orthod. Craniofac. Res. 13, 114–117.
•  Sun,  X.,  Shen,  J.,  Wu,  W.,  Xie,  J.,  Gao,  C.,  Qin,  L.,  Cui,  Y., and  Liu,  J. (2013) Identification of a novel c.822 G>T mutation of EDA gene in a Chinese family with X-linked hypohidrotic ectodermal dysplasia. Chinese journal of medical genetics 30, 270–273 (in Chinese).
•  Tape,  M. W., and  Tye,  E. (1995) Ectodermal dysplasia: literature review and a case report. Compend. Contin. Educ. Dent. 16, 524–528.
•  Vierucci,  S.,  Baccetti,  T., and  Tollaro,  I. (1994) Dental and craniofacial findings in hypohidrotic ectodermal dysplasia during the primary dentition phase. J. Clin. Pediatr. Dent. 18, 291–297.
•  Viljoen,  D. L., and  Winship,  W. S. (1988) A new form of hypohidrotic ectodermal dysplasia. Am. J. Med. Genet. 31, 25–32.
•  Wang,  H. J.,  Tang,  Z. L.,  Lin,  Z. M.,  Dai,  L. L.,  Chen,  Q., and  Yang,  Y. (2014) Recurrent splice-site mutation in MBTPS2 underlying IFAP syndrome with Olmsted syndrome-like features in a Chinese patient. Clin. Exp. Dermatol. 39, 158–161.
•  Wohlfart,  S.,  Söder,  S,  Smahi,  A., and  Schneider,  H. (2016a) A novel missense mutation in the gene EDARADD associated with an unusual phenotype of hypohidrotic ectodermal dysplasia. Am. J. Med. Genet. A 170A, 249–253.
•  Wohlfart,  S.,  Hammersen,  J., and  Schneider,  H. (2016b) Mutational spectrum in 101 patients with hypohidrotic ectodermal dysplasia and breakpoint mapping in independent cases of rare genomic rearrangements. J. Hum. Genet, 61, 891–897.
•  Yan,  M.,  Zhang,  Z.,  Brady,  J. R.,  Schilbach,  S.,  Fairbrother,  W. J., and  Dixit,  V. M. (2002) Identification of a novel death domain-containing adaptor molecule for ectodysplasin-A receptor that is mutated in crinkled mice. Curr. Biol. 12, 409–413.
•  Yang,  Y.,  Luo,  L.,  Xu,  J.,  Zhu,  P.,  Xue,  W.,  Wang,  J.,  Li,  W.,  Wang,  M.,  Cheng,  K.,  Liu,  S., et al. (2013) Novel EDA p.Ile260Ser mutation linked to non-syndromic hypodontia. J. Dent. Res. 92, 500–506.
•  Yin,  W.,  Ye,  X., and  Bian,  Z. (2013) The second deletion mutation in exon 8 of EDA gene in an XLHED pedigree. Dermatology 226, 105–110.
•  Yoshioka,  T.,  Nishikomori,  R.,  Hara,  J.,  Okada,  K.,  Hashii,  Y.,  Okafuji,  I.,  Nodomi,  S.,  Kawai,  T.,  Izawa,  K.,  Ohnishi,  H., et al. (2013) Autosomal dominant anhidrotic ectodermal dysplasia with immunodeficiency caused by a novel NFKBIA mutation, p.Ser36Tyr, presents with mild ectodermal dysplasia and non-infectious systemic inflammation. J. Clin. Immunol. 33, 1165–1174.