ARTICLE
Auteur(s) : Surasawadee Ausavarat1,2, Pranoot
Tanpaiboon3, Siraprapa Tongkobpetch1, Kanya
Suphapeetiporn1, Vorasuk Shotelersuk1
1Division of Medical Genetics and Metabolism,
Department of Pediatrics, Faculty of Medicine, Sor Kor Building
11th floor, King Chulalongkorn Memorial Hospital, Bangkok 10330,
Thailand
2Inter-Department Program of Biomedical Science,
Graduate School, Chulalongkorn University, Bangkok, Thailand
3Department of Pediatrics, Faculty of Medicine, Chiang
Mai University, Chiang Mai, Thailand
accepté le 27 Février 2008
The Conradi-Hünermann-Happle syndrome (MIM 302960), also known
as X-linked dominant chondrodysplasia punctata type 2 (CDPX2), is a
rare developmental disorder resulting from a deficiency of enzymes
involved in cholesterol biosynthesis [1, 2]. It is characterized by
skeletal dysplasia, skin and ocular abnormalities. Skin
manifestations include congenital linear, whorled or blotchy
follicular hyperkeratosis with generalized erythroderma.
Erythroderma and scaling usually resolve in the first few months of
life leaving follicular atrophoderma, variable ichthyosis,
dyspigmentation, partial alopecia and lusterless hair. Abnormal
skeletal involvement includes asymmetric limb shortening, scoliosis
and epiphyseal stippling [1-4]. The disease is usually lethal in
hemizygous males while affected females exhibit segmentally
arranged clinical features due to functional mosaicism caused by
X-inactivation [5-7].
CDPX2 is caused by mutations in the emopamil binding protein
(EBP) gene [1, 2]. The EBP gene comprises 5 exons encoding a 1.0-kb
transcript. Its protein functions as a sterol isomerase enzyme in
the cholesterol synthesis pathway [8, 9]. Nevertheless, how
depletion of this enzyme contributes to a phenotype seen in this
disorder remains elusive. In addition to the finding of abnormally
elevated levels of the cholesterol precursors, 8-dehydrocholesterol
and 8(9)-cholestenol, diagnosis of CDPX2 could be confirmed by
identification of mutations in the EBP gene. There are at least 58
different disease-causing mutations reported to date
(http://www.hgmd.cf.ac.uk, accessed January 2008).
In this study, we describe two unrelated Thai girls with CDPX2,
one being sporadic and the other being familial, with novel
mutations in the EBP gene.
Materials and methods
Clinical description
Patient 1 was a full term female infant, who was born to a healthy,
G2P1, 32-year-old mother. She was noted to have asymmetric limb
shortening and skin lesions. She had flat face with a saddle nose.
The right arm and leg were shorter than the left. She had
hyperkeratotic brownish plaques on the lower extremities following
the lines of Blaschko together with generalized brownish scales
sparing scalp, face, palms, soles, and inguinal area (figure 1A). Her hair and
nails appeared normal. At 2 months of age, she developed bilateral
cataracts. A skeletal survey performed at two weeks of age
demonstrated asymmetric shortening of the humeri and femora (figure 1B). There
were generalized punctate calcifications of the epiphyseal regions
of long bones, vertebrae and pelvic bone (figure 1C). No other
family member had similar manifestations.
Patient 2 first presented to our clinic at the age of 13 years.
She was noted to have dry and scaly skin since birth. Her left
cornea appeared cloudy at one month of age. Her mental development
was normal. Physical examination revealed a height of 122 cm
(– 4 SD). She had sparse and coarse lusterless hair with
patchy areas of alopecia. Bilateral cataracts were present. The
bridge of the nose was flat (figure 1D). She had
atrophic linear skin lesions following the lines of Blaschko mostly
on the extremities. The left upper arm and upper leg appeared
shorter than the right. Postaxial polydactyly of her left hand and
pronounced kyphoscoliosis were also present. X-ray examination of
the skeleton confirmed the clinical findings of asymmetrical
shortening of humerus and femur, postaxial polydactyly and
scoliosis (figures
1E and F).
The mother of patient 2 was 37 years old with a height of
142 cm. She had sparse hair and atrophic linear skin lesions
following the lines of Blaschko. She did not manifest cataracts,
polydactyly, scoliosis, or asymmetric limb shortening. No other
family member had similar findings. In neither family of the two
unrelated patients, was there any history of consanguinity, excess
miscarriage or male stillbirth.
Mutation analysis
After informed consent was received, 3 mL of peripheral blood
from probands and their available parents were obtained. Genomic
DNA was extracted from peripheral leukocytes according to standard
protocols. Direct cycle sequencing of PCR-amplified DNA
representing all coding exons of EBP was performed as previously
described [10]. For a novel missense mutation, restriction enzyme
digestion was used to confirm its presence in the patient and to
screen in 100 control chromosomes from unaffected
ethnically-matched individuals.
Results
PCR-sequencing analysis of the entire coding sequence of EBP
revealed that patient 1 was heterozygous for a G to T transversion
at nucleotide position 616 (c.616G→T), in exon 5 (figure 2, upper left
panel). The mutation is expected to result in an aspartic acid to
tyrosine substitution at codon 206 (p.D206Y). The mutation was
confirmed by digestion of the PCR products with the restriction
enzyme TfiI (data not shown). The c.616G→T was de novo and was not
detected in 100 ethnic-matched control X chromosomes.
Patient 2 was heterozygous for a deletion of a cytosine at
nucleotide position 382 (c.382delC) in exon 3 of the EBP gene
(figure 2, upper
right panel). The loss of a cytosine leads to a frameshift starting
at codon 128 onwards and introduces a premature stop codon at
position 137. Her mother was found to harbor the same mutation.
Discussion
We described two unrelated Thai girls who had Conradi-Hünermann-
Happle syndrome with two novel mutations in the EBP gene. Both
patients had typical manifestations of CDPX2, with one of them
having postaxial polydactyly, a rare feature of CDPX2 [3].
A single base transversion c.616G→T in exon 5 resulting in an
aspartic acid to tyrosine substitution at position 206 (p.D206Y)
was detected in patient 1. This mutation has never been previously
described and is located at the most 3’ position reported to date.
Even though no in vitro study has been performed to investigate the
functional consequence of this mutation, there are several lines of
evidence supporting it as a disease-causing mutation. First, the
variant is a non-conservative substitution, introducing a charge
difference. Aspartic acid is negatively charged while tyrosine is
uncharged. Second, the aspartic acid at codon 206 is located at a
highly conserved cytoplasmic domain. Third, PolyPhen
(http://coot.embl.de/PolyPhen) predicts it to be possibly damaging.
Fourth, this variant has not been reported to be a polymorphism in
NCBI SNP (http://www.ncbi. nlm.nih.gov/ projects/SNP), Ensembl
(http://www.ensembl.org/index.html) or PupaSUITE/PupaSNP
(http://pupasuite.bioinfo.cipf.es) databases. And lastly, it was
not detected in 100 ethnically-matched control chromosomes.
The c.382delC identified in patient 2 is also novel. This
alteration is expected to result in changing the leucine to
cysteine at position 128, subsequent changes of 8 amino acids and
truncation at amino acid 137 (p.L128CfsX137), which eliminates
almost half of the EBP protein. It is likely to result in either an
absent or nonfunctional truncated protein. Interestingly, in
addition to features commonly found in CDPX2, this patient with
c.382delC had postaxial polydactyly. This rare feature has been
previously detected in four molecularly-confirmed patients with
CDPX2 [2, 11, 12]. Two harbored different insertions (c.166-167insT
and c.268-269insCT) and the other two had nonsense mutations
(c.328C→T and c.298C→T) resulting in frameshifts and truncated
proteins, respectively.
There has been no clear evidence of a genotype-phenotype
correlation. However, it has been suggested that females with
nonsense or frameshift mutations producing a nonfunctional protein
are likely to demonstrate a more severe involvement of the disorder
while patients with missense mutations do not consistently present
all clinical features, particularly ocular abnormalities [13]. Our
two patients, one with a missense mutation and the other with a
frameshift alteration, had typical features of CDPX2 including
skeletal, skin and ocular abnormalities. The latter, with a
frameshift, also had postaxial polydactyly. No correlation,
however, could be suggested from our study.
The mother of patient 1 was asymptomatic. Nonetheless, it has
been shown that a mutation can be found in asymptomatic mothers of
sporadic cases. We, therefore, performed mutation analysis of her
parents and found that they both had only the wild-type alleles.
Cautiously, the inability to detect a mutation in the mother of a
sporadic case does not completely eliminate the risk of recurrence
for a woman who has an affected daughter, since germline mosaicism
has been reported [7]. The mother of patient 2 had clinical
manifestations but significantly milder than those of her affected
daughter. Inter- and intra-familial variations as well as
incomplete penetrance have been demonstrated [6, 7, 14]. The
expressivity of a particular mutation is likely to reflect the
pattern and timing of X-inactivation.
In summary, we reported two unrelated Thai girls with CDPX2. Two
potentially pathogenic novel mutations, c.616G→T and c.382delC were
identified. This study expands the genotypic spectrum of EBP
mutations.
Acknowledgment
We would like to thank the patients and their families for
participation in this study. This study was supported by the
Research Unit Fund, Chulalongkorn University, the National Center
for Genetic Engineering and Biotechnology, and the Thailand
Research Fund. Conflict of interest: none.
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