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A novel 4-bp insertion mutation in EDA1 gene in a Pakistani family with X-linked hypohidrotic ectodermal dysplasia

European Journal of Dermatology. Volume 17, Number 3, 209-12, May-June 2007, Genes and skin

DOI : 10.1684/ejd.2007.0150

Summary

Author(s) : Muhammad Tariq, Naveed Wasif, Muhammad Ayub, Wasim Ahmad , Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.

Summary : Hypohidrotic ectodermal dysplasia (HED) is a genetic disorder characterized by the absence or hypoplasia of hair, teeth, and eccrine sweat glands. The inheritance pattern of HED may be X-linked or autosomal (dominant or recessive). Mutations in the EDA 1 gene cause X-linked HED and mutations in either EDAR or EDARADD genes cause autosomal forms of HED. To search for a mutation in human EDA1 gene in a large Pakistani family demonstrating X-linked form of HED (XLHED), eight exons and splice junction sites of EDA1 gene were amplified by PCR from genomic DNA and sequenced directly in an ABI Prism 310 automated DNA sequencer. A novel four bases insertion mutation (913_914insTATA) was identified in exon 8 of the EDA 1 gene. This insertion introduces a reading frameshift leading to downstream premature termination codon in the same exon. In the present study a novel insertion mutation in EDA1 gene in a Pakistani family with XLHED has been reported. This extends our knowledge of mutations in EDA1 gene that define the pathogenic basis of HED.

Keywords : EDA1 gene, insertion mutation, Pakistani family, X-linked hypohidrotic ectodermal dysplasia, Christ-Siemens-Touraine syndrome

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ARTICLE

Auteur(s) : Muhammad Tariq, Naveed Wasif, Muhammad Ayub, Wasim Ahmad

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan

accepté le 3 Janvier 2007

Hypohidrotic (anhidrotic) ectodermal dysplasia (HED) (also called as Christ-Siemens-Touraine syndrome, CST syndrome) is the most common form of more than 170 related ectodermal dysplasias (EDs) [1, 2], and characterized by the absence or deficient function of at least two derivatives of the ectoderm: teeth, hair, sweat glands or nails. Various clinical forms of the disorder demonstrate different modes of inheritance. The most common variant involves X-linked inheritance, with partial manifestation in females [3, 4].X-linked hypohidrotic ectodermal dysplasia, (XLHED; MIM 305100), results in the abnormal development of teeth, hair, and ecrine sweat glands [5, 6]. XLHED is caused by mutations in the ectodysplasin gene [EDA1: ectodysplasin A1 isoform (EDA-A1); MIM 300451], located on chromosome Xq12-q13.1 [7]. EDA1 has a murine homologue tabby (Ta), found mutated in 2 independent tabby mouse strains [8]. A minority of patients with the HED (MIM 224900) phenotype display an autosomal (recessive or dominant) inheritance pattern that is due to mutations in a distinct gene, termed ectodysplasin A1 isoform (EDA-A1) receptor (EDAR; MIM 604095), located on chromosome 2q11-q13, which has dominant and recessive murine alleles termed downless (Dl and dl, respectively) [9, 10]. Furthermore, autosomal recessive inheritance in a family with HED has been found to be due to a mutation in EDAR-associated death domain (EDARADD; MIM 606603) gene (crinkled cr in mouse), located on chromosome 1q42.2-q43 [11].The EDA1 gene encodes a protein, ectodysplasin-A (EDA-A1), a member of the tumor necrosis factor (TNF) superfamily of ligands, required for the morphogenesis of hair, teeth and other ectodermal derivatives. EDA-A1 is a type II transmembrane protein with three functionally important regions in EDA-A1: a C-terminal TNF homology domain necessary for receptor binding, a bundle forming collagen domain, and a furin protease recognition sequence [12]. EDA-A1 is a ligand of EDAR, activating it and then EDAR uses EDARADD as an adapter to activate the nuclear factor (NF)-кB signaling pathway (figure 1), which contributes to ectodermal morphogenesis. This linear pathway explains the identical symptoms among HED patients displaying different modes of inheritance and the genetic heterogeneity of the disorder in mice and humans [11, 13].In the present study, we investigated a Pakistani family demonstrating X-linked HED. DNA sequence analysis identified a novel mutation in EDA1 gene, located on Xq12-q13.1.

Materials and methods

Subjects

In the present study a large Pakistani family with XLHED (figure 2) was investigated. Prior to the start of the study, approval was obtained from the Quaid-i-Azam University Institutional Review Board. Informed consent was obtained from all subjects participating in the study. All affected and normal individuals underwent examination at Department of Dermatology, Pakistan Institute of Medical Sciences, Islamabad, Pakistan. Blood samples were obtained from 15 individuals of the family including 8 males III-3, IV-2, IV-4, IV-5, IV-6, V-1, V-2, V-3 and 7 females III-4, IV-1, IV-3, IV-7, V-4, V-5, V-6 (figure 2). Genomic DNA was extracted from peripheral blood according to standard techniques [14].

Mutation analysis

Clinical features of the affected individuals and X-linked inheritance of the HED in the family demonstrated that a mutation in EDA1 gene may be responsible for the disease. Therefore, to search for an underlying mutation in EDA1 gene, exons and splice junctions of all the 8 exons were amplified by PCR from genomic DNA using primers designed from intronic sequences of the gene. PCR products were purified using the Rapid PCR Purification System 9700 (Marligen Biosciences, Ijamsville, MD, USA) and sequenced using the Big Dye Terminator Cycle Sequencing Kit (PE Applied Biosystems USA) following purification in a Centri-Spen Spin Column PE Applied Biosystems USA). To amplify 350 bp PCR fragment containing exon 8 the following primers were used.

5’-TTCTAGGCTACCCTGGTTGC-3’ (intron 7, sense).

5’-CCATTGGATGGACTTGGCTG-3’ (intron 8, antisense).

Results

Clinical findings

Affected male individuals (IV-2, IV-5, IV-6, V-1, V-3) of the family, presented here, showed the characteristic features of HED, including fine and curly sparse hair, absent eyebrows and eyelashes, conical teeth, diminished sweating, absence of axillary and pubic hair, dry and thin skin, periorbital wrinkling and hyperpigmentation, protruding prominent lips, pointed chin, frontal bossing and saddle-shaped nose (figure 3). Three female carriers IV-3, V-5 and V-6 (figure 3) exhibited significant clinical features of HED. Female carriers V-5 (figure 3D) and V- 6 have sparse hair on scalp and conical teeth. Carrier V-5 showed reduced secretion of sweat. Saddle nose and thin skin were observed in carrier IV-3. Females carriers III-3, IV-1, and V-4 had no observed features of HED and their status was determined by gene analysis. All affected males and female carriers have normal finger and toenails.

Mutation analysis

The entire coding portion and intron-exon boundaries of the EDA1 gene which were available for the study, were sequenced in all the 15 individuals of the family. Sequence analysis of exon 8 of the EDA1 gene from affected males (IV-2, IV-5, IV-6, V-1, V-3) in the family revealed a 4-bp insertion at nucleotide position 913 (913_914insTATA) (figure 4) resulting in frameshift and a premature stop codon 2-bp downstream in the same exon. This insertion was present in the heterozygous state in female carriers (III-4, IV-1, IV-3, V-4, V-5, V-6) within the family. This mutation was not found in healthy males (III-3, IV-4, V-2) and a healthy female (IV-7).

Discussion

X-linked hypohidrotic ectodermal dysplasia (XLHED) is a heritable disorder resulting from mutations in the EDA1 gene, which disrupts the morphogenesis of ectodermal structures, including hair, teeth, and ecrine sweat glands [12, 15]. The EDA1 gene product ectodysplasin-A (EDA), an epithelial morphogen, is a member of the tumor necrosis factor (TNF) family and is synthesized as a membrane-anchored precursor protein [16-18].

In XLHED males are fully affected with this disorder, however, one-third of female carriers have no obvious clinical features, another one-third have minimal findings (missing a few teeth), and a final one third have clinically significant involvement, but to lesser degree than that in affected males [19, 20]. This clinical variation among female carriers is due to the random X inactivation (Lyonization) [6]. This X-inactivation usually causes a mosaic pattern in female carriers, as manifested by the appearance of the lines of Blaschko [21]. These lines of the Blaschko are observed in many X-linked traits, such as incontinentia pigmenti, XLHED and X-linked dyskeratousis congenital. Happle [22] documented evidence photographically using the starch-iodine test, that the lines of Blaschko become manifest in the heterozygous state of various X-linked genes affecting the development or function of the skin and these lines represent a marker of the normal embryogenesis of the skin. These lines of Blaschko are evidence of the clonal proliferation of two functionally different populations of cells during early embryogenesis of the skin in female carriers with X-linked skin disorders and that these lines represent the visible consequences of Lyonization. The clinical features of affected males and female carriers observed in the present Pakistani family are similar to those reported earlier [5, 7, 23]. DNA sequence analysis in this family led to identification of a novel 4-bp insertional mutation (913_914insTATA) in exon 8 of the EDA1 gene (figure 4). This insertion mutation is a direct 4 bp tandem repeat (figure 4). Such repeats, like classical microsatellite loci, are comparatively prone to mutation by slipped strand mispairing [24]. As a result, the copy number of tandem repeats is liable to fluctuate, introducing a deletion or an insertion of one or more repeat units [25]. Frameshifting deletions or insertions result in abolition of gene expression. The mutation reported here leads to a frameshift and premature termination codon 2-bp downstream in the same exon, predicting to cause nonsense mediated decay of the mRNA or premature protein truncation [26].

According to the Human Gene Mutation Database, 2005, 85 different pathogenic mutations have been reported, mostly clustering in functionally important domains of the EDA protein: (1) TNF-homology domain, responsible for binding to receptor, mutations in which impair binding of both splice variants to their receptors; (2) collagen-like domain, indispensable for trimerization of the ligand, mutations in this domain inhibit trimerization of the TNF homology region; and (3) the consensus furin recognition site (the protease cleavage site), mutations in which prevent proteolytic cleavage of EDA [3, 12].

The insertional mutation (913_914insTATA) identified in the present study is located in the TNF homology domain, which spans the 142 amino acid region (250-391) of the EDA protein. Mutations in this domain can affect the overall structure of EDA, receptor binding site and interaction site [27].

EDA is a type II transmembrane protein with a C-terminal TNF domain, which binds to its receptor EDAR. Like most members of the TNF receptor (TNFR) family, EDA activates the NF-кB and JNK/c-fos/c-jun pathways [28, 29]. EDAR possesses an intracellular death domain which interacts with adaptor EDARDD which in turn interacts with TRAFs 1, 2, and 3 (figure 1) [11, 30]. These signaling pathways lead to cell death, proliferation or differentiation and are important in the early epithelial-mesenchymal interaction that regulates ectodermal appendage formation [5, 12, 13].

Acknowledgments

We sincerely wish to thank the family members for their participation. The work presented here was funded by the Higher Education Commission (HEC), Islamabad, Pakistan. Muhammad Tariq is supported by indigenous PhD fellowship from HEC, Islamabad, Pakistan.

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