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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