Epileptic encephalopathy in a girl with an interstitial deletion of Xp22 comprising promoter and exon 1 of the CDKL5 gene.код для вставкиСкачать
RESEARCH ARTICLE 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* 1 Service de Neurologie Pediatrique, Departement de Pediatrie, H^opital Necker Enfants Malades, AP-HP, Paris, France 2 Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), Paris, France 3 Inserm, U567, Paris, France Assistance Publique—H^opitaux de Paris, H^opital Cochin, Laboratoire de Biochimie et Genetique Moleculaire, Paris, France 4 5 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 INTRODUCTION 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: firstname.lastname@example.org Published online 19 May 2009 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/ajmg.b.30974 202 BAHI-BUISSON ET AL. [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 analysis. MATERIALS AND METHODS 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- 203 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 www.mlpa.com. 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 (www.hgvs.org). Numbering of intronic nucleotides was performed using the sequence from human contig GenBank (http://ncbi.nlm.nih.gov/entrez/). RESULTS 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 204 AMERICAN JOURNAL OF MEDICAL GENETICS PART B 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 www.interscience.wiley.com.] 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. DISCUSSION 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 BAHI-BUISSON ET AL. 205 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 (http://genome.ucsc.edu/cgi-bin/hgGateway). 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 www.interscience.wiley.com.] 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.  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, 206 AMERICAN JOURNAL OF MEDICAL GENETICS PART B 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 www.interscience.wiley.com.] 1991]. 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