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Epileptic encephalopathy in a girl with an interstitial deletion of Xp22 comprising promoter and exon 1 of the CDKL5 gene.

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Neuropsychiatric Genetics
Epileptic Encephalopathy in a Girl With an Interstitial
Deletion of Xp22 Comprising Promoter and Exon 1 of
the CDKL5 Gene
Nadia Bahi-Buisson,1,2,3 Benoit Girard,4 Agnes Gautier,5 Juliette Nectoux,2,3,4 Yann Fichou,2,3
Yoann Saillour,2,3 Karine Poirier,2,3 Jamel Chelly,2,3,4 and Thierry Bienvenu2,3,4*
Service de Neurologie Pediatrique, Departement de Pediatrie, H^opital Necker Enfants Malades, AP-HP, Paris, France
Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), Paris, France
Inserm, U567, Paris, France
Assistance Publique—H^opitaux de Paris, H^opital Cochin, Laboratoire de Biochimie et Genetique Moleculaire, Paris, France
Service de Neuropediatrie, CHU de Nantes, Nantes, France
Received 28 January 2009; Accepted 8 April 2009
We report a 2-year-old girl with early onset seizures variant of
Rett syndrome with a deletion at Xp22 detected by multiplex
ligation-dependent probe amplification (MLPA) technique.
This patient presented with tonic seizures at 7 days of life.
Subsequently, she developed infantile spasms at three months
and finally refractory myoclonic epilepsy. She demonstrated
severe encephalopathy with hypotonia, deceleration of head
growth, with eye gaze but limited eye pursuit, no language,
limited hand use, and intermittent hand stereotypies. This
combination of clinical features, suggestive of early onset variant
of Rett syndrome led us to screen the CDKL5 gene. In a first step,
screening of the whole coding sequence of the CDKL5 gene
revealed no point mutations. In a second step, we searched gross
rearrangements by MLPA and identified a microdeletion affecting both the promoter and exon 1 in CDKL5. Subsequent analysis
on a Nimblegen HD2 microarray confirmed a deletion of approximately 300 kb at Xp22, including the BEND2, SCML2, and
CDKL5 genes. In conclusion, our report suggests that searching
for large rearrangements in CDKL5 should be considered in girls
with early onset seizures and Rett-like features.
2009 Wiley-Liss, Inc.
Key words: CDKL5; MECP2; Rett syndrome; seizures; encephalopathy; microdeletion; Xp22
X-linked cyclin-dependent kinase-like 5 (CDKL5, OMIM 300203)
associated encephalopathy is a recently described X-linked disorder
with a phenotype overlapping that of Rett syndrome (RTT, OMIM
312750) and X-linked infantile spasms (ISSX, OMIM 308350)
and reminiscent of early onset seizure variant of Rett syndrome
[Hanefeld, 1985]. To date, less than 50 patients with CDKL5-related
encephalopathy have been described [Tao et al., 2004; Weaving
et al., 2004; Evans et al., 2005; Scala et al., 2005; Archer et al., 2006;
Bahi-Buisson et al., 2008a,b; Rosas-Vargas et al., 2008]. As for RTT,
2009 Wiley-Liss, Inc.
How to Cite this Article:
Bahi-Buisson N, Girard B, Gautier A, Nectoux
J, Fichou Y, Saillour Y, Poirier K, Chelly J,
Bienvenu T. 2010. Epileptic Encephalopathy
in a Girl With an Interstitial Deletion of Xp22
Comprising Promoter and Exon 1 of the
CDKL5 Gene.
Am J Med Genet Part B 153B:202–207.
CDKL5-related disorders affect almost exclusively girls, although a
few males have also been reported [Weaving et al., 2004; Elia et al.,
2008; Fichou et al., 2008]. Strikingly, these CDKL5 mutation
patients develop a suggestive three-step pattern epilepsy with very
frequent seizures and normal or subnormal interictal electroencephalogram (EEG) pattern before 3 months followed by epileptic
encephalopathy with infantile spasms in about a half, and myoclonic refractory epilepsy in a third [Buoni et al., 2006; BahiBuisson et al., 2008a]. They also show some RTT-like features such
as secondary deceleration of head growth, severe motor impairment, sleep disturbances, hand apraxia, and hand stereotypies
Grant sponsor: Institut National de la Sante et de Recherche Medicale;
Grant sponsor: ANR-Maladies Rares; Grant number: ANR-06-MRAR-003
-01; Grant sponsor: ANR E-Rare EuroRETT Network.
Nadia Bahi-Buisson and Benoit Girard contributed equally to the study.
*Correspondence to:
Thierry Bienvenu, Laboratoire de Genetique et de Physiopathologie des
Maladies Neuro-Developpementales, Institut Cochin, 24 rue du Faubourg
Saint Jacques, 75014 Paris, France. E-mail:
Published online 19 May 2009 in Wiley InterScience
DOI 10.1002/ajmg.b.30974
[Bahi-Buisson et al., 2008b] that could lead the clinician to the
molecular diagnosis.
Mutations responsible for CDKL5-related encephalopathy
are nonsense, missense, splice, or frameshift mutations, scattered
throughout the whole sequence of the gene [Tao et al., 2004;
Weaving et al., 2004; Evans et al., 2005; Scala et al., 2005; Archer
et al., 2006; Rosas-Vargas et al., 2008; Bahi-Buisson et al., 2008a,b;
Elia et al., 2008; Fichou et al., 2008]. To our knowledge, only one
case of large microdeletion removing the CDKL5 gene has been
reported in a patient with microphtalmia and microcornea, cardiac
malformation and early onset epilepsy with infantile spasms
[Van Esch et al., 2007].
We report here the first description of a 2-year-old girl with
clinical features highly suggestive of CDKL5 related encephalopathy, in whom a new genomic rearrangement deletes a part of the
BEND2 gene, the whole SCML2 gene, and both the promoter and
exon 1 in the CDKL5 gene using different molecular approaches
such as multiplex ligation-dependent probe amplification (MLPA)
technique and microarray comparative genomic hybridization
Case Report
A 2.5 year old girl was referred to our pediatric neurology center
because of refractory epilepsy and severe encephalopathy. She was
the first child of nonconsanguineous and healthy parents, born at
40.5 weeks with normal delivery. Neonatal parameters were within
normal range: birth weight, 3,420 g, 50th centile; height 48 cm, 25th
centile; head circumference 35 cm, 50th centile. Seizures started at
the age of 7 days with repeated tonic seizures with flushing of the
face, for 30 sec, five to ten times a day. On examination, diffuse
hypotonia and poor eye contact were noticed. Interictal EEG
showed normal background activity. In spite of multiple antiepileptic drugs including valproate, phenytoine, topiramate, pyridoxine, and clobazam, seizures persisted on a daily basis. At the age of
3.5 months, she developed infantile spasms in clusters, in combination with tonic–clonic seizures. Background EEG progressively
slowed with loss of physiological features and appearance of
multifocal spikes that tended to predominate on both central
regions. Her development was progressively delayed with pronounced hypotonia, progressive deceleration of head growth,
intermittent hand stereotypies and no speech development. Reevaluation at the age of 2.5 years showed a profoundly retarded girl
showing relative microcephaly (47 cm, 10th centile), limited eye
contact and acting at a developmental level of 6 months. Epilepsy
was refractory to multiple antiepileptic drugs with frequent daily
seizures consisting of a combination of spasms, massive myoclonia,
and tonic seizures. EEG showed slow background activity and
multifocal spikes. Brain MRI was normal as well as cardiac ultrasonography, extensive metabolic studies and chromosome analysis.
Based on clinical observation and epilepsy course, she fulfilled
the previously described criteria for CDKL5-related encephalopathy. MECP2 and CDKL5 point mutations and large molecular
rearrangements in the MECP2 locus were excluded by denaturing
liquid high performance chromatography (dHPLC), direct se-
quencing and MECP2 MLPA kit (MRC-Holland, Amsterdam,
NL) analysis. Conventional cytogenetic investigations were also
normal in this patient.
Molecular Investigations
Blood samples were obtained after informed consent, and the
protocol was approved by the appropriate Institutional Review
Board of the University Hospital of Cochin, France. Genomic
DNAs were extracted from peripheral whole blood samples using
standard methods. The MLPA was performed in a thermal cycler
(Applied Biosystems, Foster City, CA) using the SALSA091R Kit
from MRC-Holland. This kit provides an optimized probe mixture
for all the 21 CDKL5 exons, and 6 control fragments, of which
3 detect sequences on the X chromosome and 3 probes detect
sequences on autosomes. Information regarding the probe sequences (P189 CDKL probemix) and ligation sites can be obtained at Hybridization, ligation, and amplification were
performed as specified by the manufacturer. One microliter of the
amplification product was analyzed using an ABI 3100 automated
sequencer, with GeneScan 500 ROX (Applied Biosystems) as
the internal size standard. Data analysis was accomplished by
exporting the peak sizes, heights, and areas to an Excel file.
X-inactivation studies were also performed as previously described
[Allen et al., 1992].
Microarray Comparative Genomic Hybridization
and Deletion Breakpoint Mapping
Microarray comparative genomic hybridization analysis was
carried out using the microarray analysis platform of Nimblegen
Technologies (Roche NimbleGen, Madison, WI). Patient and
reference genomic DNA samples were independently labeled with
fluorescent dyes, co-hybridized to a NimblenGen Human CGH
2.1M (HD2) array, and scanned using a 5 mm scanner, as described
by the manufacturer. Fluorescence intensity raw data were obtained
from scanned images of the oligonucleotide tiling arrays by using
NIMBLESCAN 2.5 extraction software (Roche Nimblegen, Madison, WI). For each spot on the array, log 2 ratios of the Cy3-labeled
test sample versus Cy-5 reference sample were calculated. Walking
PCR was then used to locate the breakpoint. Briefly, a series of
primer pairs designed from the genomic sequence both upstream
and downstream of the suspected deletion (i.e., BEND2 gene and
intron 1 of CDKL5, respectively) were studied. PCR amplification
was performed using 30 ng of DNA from the patient. When the
primer pair is outside of the deletion, a PCR product is detected
subsequently purified and directly sequenced using the BigDye
Terminator v.3.1 Cycle sequencing kit (Applied Biosystems). The
detected rearrangement was named according to the nomenclature
recommendations ( Numbering of intronic
nucleotides was performed using the sequence from human contig
GenBank (
The MLPA assay detected a decreased copy number of exon 1 in the
CDKL5 gene (Fig. 1, two-fold decrease) while all other exons were
FIG. 1. Deletion of exon 1 in the CDKL5 gene revealed by direct comparison of the MLPA electrophoresis peak patterns (patient RTT783 in blue and
female control in red). Probe with reduced signal is indicated by an arrow. [Color figure can be viewed in the online issue, which is available at]
not affected, suggesting that the patient has a deletion and that the
downstream breakpoint is localized between exon 1 and exon 2 of
the CDKL5 gene (NM_003159). Previous studies have shown that
high-density oligonucleotide array CGH technology is an efficient
tool to detect deletions as short as few kilobases pairs [Saillour et al.,
2008]. We employed this technology to finely map the Xp22
deletion in this patient. Fine mapping of deletion breakpoints by
comparative genomic hybridization using the NimbleGen Human
CGH 2.1M whole-genome tiling v2.0D array showed that the
telomeric breakpoint locates very close to the BEND2 gene
(genomic position X:18,109,731), and that the centromeric breakpoint is located between exon 1b (present in the alternative transcriptional splice variant NM_001037343) and exon 2 in the CDKL5
gene (X:18,391,916) (Fig. 2). Our results suggest that we detected a
deletion of approximately 300 kb at Xp22, extending from the
BEND2 gene to intron 1 of the CDKL5 gene. Parents were found not
to carry this deletion, suggesting a de novo event. The deleted region
harbors only three genes, BEND2 (formerly known as CXorf20),
SCML2, and CDKL5. Then, the end-points of this deletion were
determined at the genomic level using long-range PCR. Primer
pairs, specifically positioned in predicted flanking regions of
BEND2 and exon 2 of the CDKL5 gene, were used to span and to
sequence the breakpoint. Sequencing showed normal intron 6 of the
BEND2 gene sequence up to nucleotide position X:18,109,066
(NM_153346.3; BEND2, c.1016-375) followed by CDKL5
intron 1 sequence beginning at nucleotide position X:18,407,008
(NM_003159.2; CDKL5, c.-162-27968) (Fig. 3). Comparative analysis of these genomic regions showed high sequence homology
between BEND2 intron 6 and CDKL5 intron 1 (Fig. 3, lower panel,
89% similarity over a homologous 103-bp region), suggesting that
the deletion involves perfect repeats of 27 bp at its breakpoints as
previously shown for other deletions. Therefore this deletion spans
297.94 kb, and a total of three genes (BEND2, SCML2, and CDKL5)
were directly affected by the aberration, and all were very likely to be
inactivated and to produce no transcript from the X chromosome.
In this report, a submicroscopic microdeletion of 300 kb at Xp22,
encompassing the promoter region and the exon 1 of the CDKL5
gene was identified in a girl with early onset epileptic encephalopathy reminiscent of CDKL5-related disorder. Clinical features of the
present case were highly suggestive of CDKL5-related disorder, with
early onset epilepsy evolving into epileptic encephalopathy with
infantile spasms [Bahi-Buisson et al., 2008a]. In combination with
such severe epilepsy, our patient also showed secondary deceleration of head growth, severe motor impairment, hand apraxia, and
stereotypies [Bahi-Buisson et al., 2008b]. With such a clinical
suspicion, CDKL5 mutation analysis was performed, and led to
the identification of a new microdeletion of approximately 300 kb at
Xp22. This microdeletion interrupted the BEND2 and CDKL5
genes and deleted the whole SCML2 gene. This former gene shares
high homology with the Drosophila Polycomb group (PcG) of genes
that encodes transcription factors involved in the transcriptional
repression of HOX genes, which are key factors during embryonic
development [Van de Vosse et al., 1998]. The BEND2 gene encodes
FIG. 2. Deletion mapped by oligonucleotide array CGH in the girl with severe epileptic encephalopathy. The CGH results show the log 2 intensity ratios
of the patient versus reference DNA on the vertical axis (upper panel). Each individual probe is represented by a single dot and the horizontal axis
shows the position of each probe along the X chromosome. The horizontal arrow indicates a cluster of dots located between 0.5 and 1.0 (score
0.553) that identifies the deletion. An idiogram of chromosome X is represented below ( Genes
residing in this deleted genomic region are shown below the deletion map. [Color figure can be viewed in the online issue, which is available at]
for a protein (799 amino acids) of unknown function containing an
NLS (nuclear localization site) domain. As the patient described in
this study had a typical mutated CDKL5 pattern, we suggest that
heterozygous deletion of SCML2 and truncation of the BEND2 gene
were associated with no apparent additional phenotype. To our
knowledge, this is the first description of a female patient with a
small microdeletion at Xp22 deleting the promoter region and exon
1 of the CDKL5 gene. Interestingly, Van Esch et al. [2007] reported a
patient with early onset infantile spasms combined with bilateral
microphtalmia with microcornea and tetralogy of Fallot in whom
they found an interstitial deletion at Xp22.2-Xp22.13. This deletion
of 2.8 Mb was larger than our new microdeletion, and included 16
genes or transcripts. Among the deleted genes, two candidates were
suspected to account for the phenotype, respectively the NHS gene
for bilateral microphtalmia with microcornea, and the CDKL5 gene
for early onset infantile spasms. Deletion of both SCLM1, SCLM2,
and RAI2 have been suggested to cause tetralogy of fallot [Van Esch
et al., 2007]. Remarkably, our patient does not show either a cardiac
defect or ophthalmologic disturbance, suggesting that SCLM2 and
BEND2 deficiencies do not necessarily result in multiple congenital
malformation as previously suggested [Van Esch et al., 2007].
Our deletion detected by MLPA, and confirmed by CGH array,
was also clearly defined by PCR. It appears to involve direct repeats
of 27 bp (100% identity). The presence of such short sequence
homologies at the breakpoints of large deletions is well documented
in a number of human disease genes [Kornreich et al., 1990;
Audrezet et al., 2004]. These deletions are thought to result from
slipped mispairing during DNA replication [Krawczak and Cooper,
FIG. 3. Direct sequencing of the junction fragment obtained by PCR from genomic DNA of the heterozygous patient for the deletion. Sequence
alignment of intron 1 of the CDKL5 gene and intron 6 of the BEND2 gene showing the homology region of about 100 base pairs around the
breakpoints. The 27 base pairs direct repeats are in bold. Identity between the two genomic sequences is indicated by a short vertical line. [Color
figure can be viewed in the online issue, which is available at]
1991]. As it has been demonstrated, at least in prokaryotes, that the
frequency of slipped mispairing is proportional to both the length
of the direct repeat motif and the extent of homology between the
direct repeats, but inversely proportional to the distance between
them [Krawczak and Cooper, 1991], we can suppose that this kind
of deletion (similar or identical) may be recurrent. In conclusion,
our data suggest that the screening of large rearrangements, such
as large CDKL5 deletion at the heterozygous state that are undetected using current PCR-based techniques, should be performed in
girls compatible with the CDKL5 disease profile [Bahi-Buisson
et al., 2008b] using MLPA, real-time quantitative PCR, or CGH
This work was supported by Institut National de la Sante et de
Recherche Medicale (ANR-Maladies Rares ANR-06-MRAR-00301, and ANR E-Rare EuroRETT Network).
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cdkl, promote, epileptic, exon, encephalopathy, interstitial, comprising, xp22, girl, genes, deletion
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