ARTICLE
Auteur(s) : Susan C Kelly1,
Paulina Ratajczak2, Matthew Keller2, Stephen
M Purcell1, Thomas Griffin1, Gabriele
Richard2
1Department of Dermatology, Lehigh Valley Hospital,
Allentown, Pennsylvania, USA
2Department of Dermatology & Cutaneous Biology and
Institute of Molecular Medicine, Thomas Jefferson University,
Philadelphia, Pennsylvania, USA
accepté le 23 Janvier 2006
Oculo-dento-digital dysplasia (ODDD) is a rare developmental
multisystemic disorder first described by Lohmann in 1920 [1].
Approximately 300 patients have been reported [2-12]. The major
clinical features include: 1) facial dysmorphism with a thin nose
and hypoplastic alae, 2) eye findings, such as microphthalmia, and
glaucoma, 3) syndactyly, camptodactyly (permanent joint flexion) of
the digits, 4) teeth anomalies, enamel hypoplasia, and 5) heart
defects. Less common ancillary features are epicanthal folds and
neurological symptoms [2, 6-8]. In recent years, it has been
recognized that a number of ODDD patients also have abnormalities
of the skin and its appendages, such as brittle nails [8], slowly
growing hair, and sparse [7, 9-11], curly or kinky [2] hair and,
very rarely, palmoplantar keratoderma [8, 9]. Considering these
cutaneous features and all the other manifestations, ODDD can be
classified as an ectodermal dysplasia (ED) with widespread system
involvement.ODDD is an autosomal dominant disorder with
considerable phenotypic variability. Advanced paternal age has been
noted in some sporadic cases [7]. Heterozygous mutations in the
connexin 43 gene (GJA1) on chromosome 6q22-q24 have been reported
in 26 unrelated ODDD patients and families to date [3, 9, 12] These
mutations are thought to exert a dominant negative effect and
interfere with normal coupling of cells via gap junctions
[13].Disruption of Cx43-mediated cell to cell communication, may
lead to disruption in the morphological patterning during
development and alter the function of cells in mature tissue [14].
The gap junction protein Cx43 is almost ubiquitously expressed in
various tissues of ectodermal and mesodermal origin, which might
explain the complex phenotype and widespread organ involvement in
ODDD. We present here a patient with ODDD and multiple cutaneous
features due to a novel missense mutation in GJA1.
Materials and methods
Patients and biological material
We ascertained a 13 year-old girl with ODDD (II-4), her 3
unaffected siblings and both unaffected parents (figure 2A). DNA was
extracted from buccal swabs or venous blood samples following
standard procedures. The studies were approved by the institutional
review board and performed with informed consent of all
participants.
DNA amplification and mutation analysis
The coding region of GJA1 and flanking intronic and 3’UTR sequences
were PCR amplified from genomic DNA samples in 3 overlapping
fragments as previously described [15], gel purified and subjected
to bidirectional DNA sequence analysis. The mutation was confirmed
by bi-directional DNA sequencing using primers 5’-AGA AAT ACG TGA
AAC CGT TGG-3’ (forward) and 5’-TGT CCA CAT TGA CAC CA-3’
(reverse), as well as by denaturing high performance liquid
chromatography (dHPLC). This method was also used to exclude the
mutation from 120 population-matched control chromosomes. For dHPLC
analysis, a 379 bp fragment of GJA1 was PCR-amplified from genomic
DNA with primers 5′-GGG TGA CTG GAG CGC CT-3′ (forward) and 5′-CAA
GTG CAT GTC CAC ATT GA-3′ (reverse). Each sample was mixed with a
wildtype control sample in a ratio of 2:1 (vol/vol), denatured at
94 °C for 10 min, cooled to 65 °C for 10 min to
allow heteroduplex formation and maintained on ice until loading.
Ten μl of each sample were separately injected and analyzed on a
WAVE™ DNA fragment analysis system (Transgenomic Inc., Omaha, NE),
over 7.5 min at a temperature gradient of 59 °C that was
established with the WAVE Maker program 3.4 (Transgenomic Inc.)
Paternity was confirmed by genotyping 10 polymorphic loci on
different chromosomes using the PowerPlex® 16 system
(Promega, Madison WI) and their recommended conditions.
Electronic database information.
http://www3.ncbi.nlm.nih.gov/Omim/ searchomim.html;
http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html;
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide;
http://searchlauncher.bcm.tmc.edu/multi-align/Options/clustalw.html
http://www.promega.com/techserv/apps/hmnid/referenceinformation/popstat/custstat_Allelefreq.htm
(population-specific allele frequencies for PowerPlex®
16 system markers).
Results
Clinical features
A 13-year-old female was born full-term to non-consanguineous
parents of Northern European origin, who were in their early
thirties. She had epicanthal folds, a narrow nose, high-arched
palate and bilateral webbing of her 4th and
5th fingers, which was surgically repaired during
infancy. At 4 months of age she was diagnosed with glaucoma. She
had supernumerary teeth (figure 1A) and a cleft
palate necessitating oral surgery and notably yellow discoloration
of the primary and permanent teeth. Her scalp hair had always been
short, curly, and slow growing.
The patient’s medical history was negative for cardiac
disorders, neurologic symptoms, mental deficiency, deafness, or
sweating abnormalities. There were no family members with similar
facies, curly/kinky hair, dental or skeletal abnormalities.
Medications were limited to topical glaucoma therapy.
Physical examination revealed a beak-like nose with hypoplasia
of the alae nasi, anteverted nostrils, and prominent columella. The
patient had microphthalmia, hypotelorism, and prominent medial
epicanthal folds with focal scars (figure 1B). Scalp hair was
short, with a curly-kinky texture and there was marked hypoplasia
of the dental enamel with yellow-colored teeth (figure 1C). Bilateral
4th and 5th fingers demonstrated interdigital
scarring from syndactyly repair and radial camptodactly (figure 1D). Discrete
follicular hyperkeratosis was noted on the extensor surfaces of the
extremities and she had mild palmoplantar keratoderma. There were a
few well-demarcated hyperkeratotic, mildly scaly plaques on the
anterior wrists and ankles (figure 1E).
On light microscopic examination, scalp hairs appeared short and
fine, with irregular light and dark brown pigmentation of the hair
shafts. A small portion of the hair shafts had a beaded appearance
with flat, wide nodules interrupting the hair at irregular
intervals. Rare beaded areas demonstrated subtle peripheral
trichorrhexis (figure
1F) consistent with early trichorrhexis nodosa or
pseudomonilethrix [16]. Scanning electron microscopy (figure 1G) revealed focal,
semi-smooth, ovoid dells in the beaded areas likely representing
weakened areas of the hair shaft.
Mutation analysis reveals a novel heterozygous missense
mutation in GJA1 (Cx43)
Coding and flanking sequences of the GJA1 (Cx43) gene were screened
for the presence of sequence variants in the patient’s genomic DNA.
Sequence analysis revealed a heterozygous T->C transition at
nucleotide 32 (from ATG start site) in GJA1 (figure 2B). This point
mutation can be predicted to lead to a non-conservative replacement
of leucine 11 (CTT) with a proline residue (CCT)
(L11P) in the cytoplasmic amino terminus of Cx43. In fact, leucine
is invariably present at this position in all human Cx genes with
exception of GJB6 (Cx30), which has an isoleucine at this position
(figure 2C).
Since proline is an imino acid that does not form hydrogen bonds
and has restricted conformational space, the substitution of
proline for leucine might have significant consequences on the
high-order structure of Cx43. As demonstrated by sequence analysis
or dHPLC, no other first degree relative harbored this missense
mutation. Paternity was confirmed in this nuclear family with a
probability of 0.999999286 by genotyping 10 microsatellite markers
with known allele frequency in the White US population. Mutation
L11P was also not detected in 120 chromosomes of unaffected
individuals of Northern European origin (figure 2A). These findings
exclude the possibility that L11P represents a non-consequential
sequence polymorphism and support the pathogenicity of this
mutation. No sequence aberrations were found in the remainder of
the coding sequence of GJA1 or in the Cx genes GJB2, GJB3, GJB4 and
GJB6.
Discussion
Oculodentodigital dysplasia (ODDD) is a disorder with distinct
clinical features affecting both ectodermal and mesodermal cell
lineages. The developmental abnormalities in ODDD appear to be
caused by impaired Cx43 function during embryogenesis and
epithelial differentiation. Cx43 belongs to a family of 21 closely
related gap junction proteins, which form intercellular
’communication’ channels and thereby permit regulatory molecules,
ions and small nutrients to freely pass between cells. These gap
junction channels are vital for the control and coordination of
many physiologic and developmental processes in human tissues.
Recent studies of gene-targeted animal models have fostered our
understanding and appreciation for the role of Cx43 during
organogenesis.
Cx43 is expressed in various tissues such as brain, heart,
gonads, lens, cornea, skin and bone [17]. In chicks, high levels of
Cx43 were found to be expressed in the cornea, lens, and neural
retina as early as days 2-4 of embryogenesis and required for the
control of early neurogenesis and eye formation [18]. Reduction of
Cx43 expression in this animal model resulted in microphthalmia and
a smaller retina [18]. These data correspond well with the ocular
abnormalities in ODDD, including cataracts, optic atrophy,
microcornea, as well as microphthalmia and glaucoma as seen in our
patient. In the postnatal tooth development of rats, Cx43 channels
have been demonstrated between the odontoblasts and appeared to be
necessary for the secretion of the dentin matrix [19]. Deletion of
Cx43 in murine models has resulted in osteoblast dysfunction,
delayed mineralization, decreased bone mass and skeletal
abnormalities [20, 21]. Interestingly, Cx43 is richly expressed in
the human myocardium, where cardiac myocytes are electrically
coupled via gap junctions. These gap junctions are responsible for
the coordinated electrical impulse conduction, which allows the
heart to function as a syncytium [22]. Together, these observations
could explain the dental, skeletal and cardiac abnormalities in
ODDD due to dominant negative mutations in the Cx43 gene.
In epithelia of ectodermal origin, Cx43 is abundantly expressed
in the lower portion of the hair shaft, the cortex, inner and outer
root sheath as well as the interfollicular areas of the skin [23,
24]. Therefore it is not surprising that skin and hair shaft
abnormalities may occur in ODDD, but only a few case reports have
drawn attention to these findings [2, 7-11, 25]. In our patient, we
identified curly-kinky hair with features of early trichorrhexis
nodosa. This is consistent with the observation by Kjaer et al.,
who found curly hair in 7 out of 9 ODDD patients harboring a
mutation in the Cx43 gene [2].
Cx43 is also the most prevalent gap junction protein of the
epidermis, expressed throughout all layers [23, 24, 26, 27].
However, its expression widely overlaps with a battery of other
connexins, such as Cx31, Cx30.3, Cx40, Cx45, and Cx30. Germline
mutations in many of these gap junction proteins have been found to
cause inherited skin disorders. Autosomal dominant missense
mutations in the connexin 26 gene (GBJ2) underlie a spectrum of
syndromic skin and hearing disorders with overlapping features,
including KID (keratitis-ichthyosis-deafness) syndrome,
palmoplantar keratoderma associated with sensorineural hearing
loss, Vohwinkel syndrome and Bart-Pumphrey syndrome. Mutation in
the connexin 30 gene (GJB6) may cause Clouston syndrome (hidrotic
ectodermal dysplasia) or KID syndrome with atrichia, while
mutations in the Cx31 or Cx30.3 genes (GJB3, GJB4) are responsible
for erythrokeratodermia variabilis [28]. Many of these cutaneous
connexin disorders involve multiple tissues of ectodermal origin,
similar to ODDD, and thus have been categorized as ectodermal
dysplasias (ED).
To date, 26 out of 27 Cx43 mutations linked to ODDD are
clustered in the N-terminal two-thirds of the Cx43 amino acid
sequence. Paznekas et al identified 17 GJA1 mutations in ODDD
patients [8] and in 2004 Richardson et al. identified 10 additional
mutations in GJA1, seven of which were novel [3]. Kellermayer et
al. (2005) reported a patient with hidrotic ectodermal dysplasia
and extensive hyperkeratosis of the skin, who also had abortive
features of oculo-dentodigital dysplasia [15]. Interestingly, this
patient harbored a novel mutation in GJA1 (V41L) as well as a
heterozygous sequence variant of GJB2 (R127H), suggesting that the
clinical expressivity of GJA1 mutations may be modified by sequence
variants in other connexin genes. In 2005, Vasconcellos and
colleagues identified a novel proline to histidine mutation (P59H)
in GJA1 [12]. Van Steensel et al. also reported in 2005 a 2-bp
deletion in the GJA1 gene, the only ODDD mutation noted in the
C-terminus of Cx43, and the authors hypothesized that the unique
nature and location of this mutation could explain the prominent
skin involvement in their case [9]. Nevertheless, the mutation
reported here associated with kinky hair, early trichorrhexis
nodosa and keratoderma falls within the cytoplasmic N-terminus
where other ODDD mutations without skin features have been
reported. Recent in vitro expression studies of Cx43 harboring a
nearby misssense mutation, Y17S, in the N-terminus demonstrated a
complete absence of gap junction intercellular communication
between apposing N2A cells despite the normal formation of gap
junction plaques by the mutant protein [13]. These data suggest
that N- terminal Cx43 mutation have a detrimental effect on channel
conformation and gating which has also been observed for mutations
in Cx32 [28].
As illustrated in this report, it is important for
dermatologists to recognize ODDD as an ED, which can present with
hair and nail abnormalities and epidermal hyperkeratosis. Typically
EDs have been classified solely on clinical grounds [29]. However,
Priolo et al. contend that EDs are “not only skin deep” and
proposed a new classification of EDs [30]. Integrating
molecular-genetic data with the corresponding clinical findings,
they proposed to group EDs based on the function of the defective
proteins and underlying pathomechanisms. A relatively similar
concept was also suggested by Lamartine in 2003 and supported by
Itin in 2004 [31, 32]. EDs were classified into 4 functional
groups: cell-cell communication and signaling; cell adhesion;
transcription regulation; and development [31]. However, as vividly
illustrated by the example of ODDD, these schemes are marred by the
fact that many proteins fulfill multiple functions in adult tissues
or during development. While ODDD presents the diagnostic criteria
of an ED with involvement of multiple ectodermal and mesodermal
tissues, the disorder obviously crosses the lines between the
categories of cell-cell communication/signaling and developmental
function.
To date, 27 different GJA1 mutations have been reported in
association with ODDD, including the novel mutation reported here.
Abnormalities of the skin, hair, and nails are not unusual in ODDD
but are easily overlooked. We have identified a novel de novo
missense mutation in Cx43 (GJA1) gene in our patient with ODDD with
curly hair, early trichorrhexis nodosa and discrete keratoderma. In
light of these cutaneous findings and based on recent ED
classification systems we propose to include ODDD in the group of
ED.
Acknowledgements
We would like to thank the family for their participation. This
work was funded in part by the National Foundation for Ectodermal
Dysplasia, the American Skin Association, the Dermatology
Foundation, the Foundation for Ichthyosis and Related Skin Types
and NIAMS/NIH grants K08-AR2141 and P01-AR38923 (GR).
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