- Email: [email protected]
Somatic mosaicism for Y120X mutation in the MECP2 gene causes atypical Rett syndrome in a male
Brain & Development 33 (2011) 608–611 www.elsevier.com/locate/braindev
Case report
Somatic mosaicism for Y120X mutation in the MECP2 gene causes atypical Rett syndrome in a male Juan I Pieras a,b, Beatriz Mun˜oz-cabello c, Salud Borrego a,b, Irene Marcos a,b, Javier Sanchez a,b, Marcos Madruga c, Guillermo Antin˜olo a,b,⇑ a
Unidad de Gestio´n Clı´nica de Gene´tica, Reproduccio´n y Medicina Fetal, Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocı´o/CSIC/Universidad de Sevilla, Spain b Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras (CIBERER), Spain c Unidad de Gestio´n Clı´nica de Pediatrı´a, Seccio´n de Neuropediatrı´a, Hospital Universitario Virgen del Rocı´o, Spain Received 17 May 2010; received in revised form 24 September 2010; accepted 27 September 2010
Abstract Rett Syndrome (RS; MIM_312750) is a severe and progressive neurodevelopmental disorder affecting principally females. Mutations in X-Linked MECP2 gene (methyl CpG-binding protein 2; MIM_300005) have been reported as being the major cause of RS. Mutations in this gene have been described as cause of wide spectrum of neurological disorders and mental retardation in males. In some cases, mutations in MECP2 in males produce clinical picture similar to RS. Here we report the identification of the novel truncating mutation Y120X in a 4-year-old child with atypical RS phenotype. Chromosome analysis showed a normal karyotype, and blood DNA and tissue DNA analysis reveal a mosaic for the mutation. Patient’s mother DNA analysis showed that this is a de novo mutation, that has never been described before in any female or male case of RS. Ó 2010 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved. Keywords: Rett Syndrome; MECP2; Somatic mosaicism; Male patient
1. Introduction Rett syndrome (RS) is a X-linked genetic disease and one of the most common causes of genetic mental retardation in females [1]. MECP2 gene (methyl CpGbinding protein (2) was associated to RS due to identification of mutations in this gene in more than 90% of RS females [2] and 1.3–17% in RS males [3,4]. RS is a progressive neurodevelopmental disorder characterized by a
⇑ Corresponding author at: Unidad de Gestio´n Clı´nica de Gene´tica, Reproduccio´n y Medicina Fetal, Hospital Universitario Virgen del Rocı´o, Avenida Manuel Siurot s/n, 41013 Seville, Spain. Tel.: +34 955 012 772. E-mail address: [email protected] (G. Antin˜olo).
normal postnatal development until 6–18 months of age, followed by stagnation and regression of acquired skills and purposeful movement of the hands. Patients develop a severe disorder with secondary microcephaly, central respiratory deregulation, stereotypic hands movements and often have intractable seizures. Initially, MECP2 mutations in males were considered lethal, nevertheless, in the last decade several mutations have been described in males affected with a wide spectrum of neurological disorders, such as severe encephalopathy, Angelman syndrome-like phenotype [5] or X-Linked mental retardation [6]. Furthermore, several male cases with a classical RS phenotype have been described associated to X-Chromosome aneuploidy, or somatic mutation [7]. We report a novel mosaic mutation p.Y120X in a male patient with atypical RS phenotype.
0387-7604/$ - see front matter Ó 2010 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2010.09.012
J.I Pieras et al. / Brain & Development 33 (2011) 608–611
2. Patients and methods 2.1. Case report The patient was the first child born from healthy nonconsanguineous parents. At birth, weight was 3900 grs, head circumference was 35 cm and Apgar score was 9 at 1 minute and 10 at 5 minutes. During neonatal period, the child presented global hypotonia and congenital torticollis. At 7 months, he presented normal head circumference, no dysmorphic features and developmental delay with axial hypotonia, mild distal spasticity, reduced global motility and no head control. Visual contact was absent and the patient did not show any interest for environmental stimuli. He had no purposeful use of hands and presented bimanual and oromanual stereotypes, as well as rocking stereotypes. At 2-years-old, the boy presented tonic generalized epileptic seizures, episodes of unmotivated laughing and teeth grinding. Later on, he presented other tonic seizures and partial seizures. At 3-years-old, the patient associated myoclonic jerks provoked by sonorous stimuli, had achieved sedestation, was able to stand up and was starting unstable and ataxic walking with support. He continued with no purposeful use of hands, as well as oromanual and bimanual stereotypes. At this moment, intense visual contact was present, but language was absent and the child presented sleep disorders. Cranial circumference was 50 cm (P50). Cranial MRI, electromyography, electroneurography and screening of metabolic diseases presented normal results. Electroencephalography (EEG) performed when he suffered the first seizures showed epileptic abnormalities with slow spike-wave complex. The clinical picture of continuous manual stereotypes, lack of purposeful use of hands and intense eye contact raised the suspicion of atypical RS. 3. Methods Genomic DNA from the patient and his mother was obtained from blood by standard procedures. In addition, we obtained DNA extracted from patient’s skin sample. An informed consent was obtained from all the participants. The study conformed to the tenets of the declaration of Helsinki and was approved by the “Committee of Ethics and Clinical Investigation” from Hospital Universitario Virgen del Rocı´o. The coding region of MECP2 gene was analyzed by direct sequencing using the Big-Dye Terminator Kit v3.1 and ABI3730 DNA analyzer (Applied Biosystems). Standard peripheral blood cultures were set up. Metaphases were prepared from 72-h culture and 20 G-banded metaphases were analyzed. FISH studies were performed using the protocol provided by the manufacturer (Vysis, Downer, Grove, IL), using chromosome X and Y centromeric probe and evaluating a total of 200 nuclei. To confirm
609
the results chromosomal and FISH analysis were performed on cultured fibroblasts from skin biopsy. MLPA method was employed, using MLPA-P015 kit, purchased from MRC-Holland (Amsterdam, The Netherlands), and according to manufacturer’s recommendations. This MLPA kit contains probes for each of the four exons of MECP2 gene and is designed to detect deletions/duplications. Fragment analyzes were performed using the 3730 DNA analyzer (Applied Biosystems) and for data analysis we used GeneMarker v 1.6 (Softgenetics L.L.C). We normalized the samples by the peak height, and included control individuals who had previously been confirmed to have no CNVs of the studied genes. Exon 3 of the MECP2 gene was PCR amplified from DNA derived from tissue and blood sample, and PCR fragments were subcloned into pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA). 20 clones from each one were then sequenced using the Big-Dye Terminator Kit v3.1 and analyzed on a 3730 sequencer. Finally, we performed a Clustawl analysis, in order to address the interspecies conservation of the methyl-CpG-binding domain (MBD). 4. Results The chromosome analysis from peripheral blood revealed a normal karyotype 46, XY. The same result was observed following karyotype analysis and FISH on cultured fibroblasts from skin biopsy. MLPA studies from blood DNA, revealed a normal gene dosage for MECP2. MECP2 gene sequence analysis revealed the presence of the variant c.360T > G together the wild-type c.360T (NM_015845) in blood DNA. The presence of these 2 variants was confirmed by gene sequencing from DNA obtained from tissue sample (Fig. 1). The analysis of 40 clones with PCR fragments of exon 3 of the MECP2 derived from tissue and blood sample respectively, revealed the presence of the mutated and the normal alleles in different clones, with a ratio of 3/20 and 2/20 (Supplementary Figure). The DNA analyzes of his mother showed no sequence alteration, revealing a de novo origin. The c.360T > G mutation causes a premature stop codon at 120 amino acid (Y120X) within the MBD of MECP2 protein. This region is a specific domain of 72 residues composed of the amino acids between 102–174 positions according to ScanProSite prediction. The Clustawl analysis of these 72 residues, MBD, revealed a highly interspecies conservation. The existence of the variant c.360T > G together with the wild-type c.360T in both blood and tissue samples, and the presence of both variants separated in different clones are indicating somatic mosaicism in our RS patient. Also, the presence of a normal karyotype, and
610
J.I Pieras et al. / Brain & Development 33 (2011) 608–611
Fig. 1. MECP2 sequence electropherograms of blood DNA obtained of the patient (1) tissue DNA obtained of the patient (2) and blood DNA obtained of his mother (3). Mutation c.360 T > G, (p.Y120X), are boxed.
the absence of MECP2 duplications are consistent with this hypothesis. 5. Discussion MECP2 is a silencing gene that controls timing for the inactivation of several functional genes in the perinatal period. Rett Syndrome (RS; MIM_312750) is a severe and progressive neurodevelopmental disorder affecting principally females [8,9]. In the last years, three groups of male patients with mutations in MECP2 gene have been defined. First group is composed of patients who have mutations in MECP2 gene identified previously in classical RS females. Those patients have severe neonatal encephalopathy and usually die within 1–2 years after birth. In addition, It has been identified a subgroup of males with normal life span and typical RS picture. These patients usually present a XXY-karyotype or a somatic mosaicism for the mutation. The second group of male patient’s present mutations not previously described in RS females. These mutations are compatible with adult life and present a wide range of mental retardation. The third group is composed of males having a duplication of the whole MECP2 gene. They present a severe clinical picture, including infantile hypotonia, recurrent respiratory infection, severe mental retarda-
tion, absence of speech development, seizures and spasticity [4]. In this paper, we report the identification of a novel de novo mutation in the MECP2 detected in a 3 yearsold boy. The mutation, namely, Y120X, has not been described before in any female case. The new truncating variation Y120X, is located in the middle of MECP2 functional domain and would produce a truncating protein with an incomplete MBD. In contrast to other RS males with a MECP2-mutation and XXY-karyotype or somatic mosaicism with typical RS picture, that is normally seen in female patients, our patient presents atypical RS phenotype. Although it would be necessary further functional analysis, the alteration of this MBD domain could affect the protein function resulting in atypical RS phenotype in this patient. Thus, RS features and survival of this patient could be explained by the existence of somatic mosaicism.
Acknowledgements We would like to thank the family who participated in the study. The CIBER de Enfermedades Raras is an initiative of the ISCIII. J.I.P. was supported by Fondo de Investigacio´n Sanitaria.
J.I Pieras et al. / Brain & Development 33 (2011) 608–611
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.braindev.2010.09.012. References [1] Ben Zeev Ghidoni B. Rett syndrome. Child Adolesc Psychiatr Clin N Am 2007;16:723–4. [2] Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 1999;23:185–8. [3] Meloni I, Bruttini M, Longo I, Mari F, Rizzolio F, D’Adamo P, et al. A mutation in the rett syndrome gene, MECP2, causes X-linked mental retardation and progressive spasticity in males. Am J Hum Genet 2000;67:982–5.
611
[4] Villard L. MECP2 mutations in males. J Med Genet 2007;44:417–23. [5] Watson P, Black G, Ramsden S, Barrow M, Super M, Kerr B, et al. Angelman syndrome phenotype associated with mutations in MECP2, a gene encoding a methyl CpG binding protein. J Med Genet 2001;38:224–8. [6] Ju¨lich K, Horn D, Burfeind P, Erler T, Auber B. A novel MECP2 mutation in a boy with neonatal encephalopathy and facial dysmorphism. J Pediatr 2009;155:140–3. [7] Topcßu M, Akyerli C, Sayi A, To¨ru¨ner GA, Kocßog˘lu SR, Cimbisß M, et al. Somatic mosaicism for a MECP2 mutation associated with classic Rett syndrome in a boy. Eur J Hum Genet 2002;10:77–81. [8] Matijevic T, Knezevic J, Slavica M, Pavelic J. Rett syndrome: from the gene to the disease. Eur Neurol 2009;61:3–10. [9] Hagberg B, Hanefeld F, Percy A, Skjeldal O. An update on clinically applicable diagnostic criteria in Rett syndrome. Comments to Rett Syndrome Clinical Criteria Consensus Panel Satellite to European Paediatric Neurology Society Meeting, Baden Baden, Germany, 11 September 2001. Eur J Paediatr Neurol 2002;6:293–7.
Case report
Somatic mosaicism for Y120X mutation in the MECP2 gene causes atypical Rett syndrome in a male Juan I Pieras a,b, Beatriz Mun˜oz-cabello c, Salud Borrego a,b, Irene Marcos a,b, Javier Sanchez a,b, Marcos Madruga c, Guillermo Antin˜olo a,b,⇑ a
Unidad de Gestio´n Clı´nica de Gene´tica, Reproduccio´n y Medicina Fetal, Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocı´o/CSIC/Universidad de Sevilla, Spain b Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras (CIBERER), Spain c Unidad de Gestio´n Clı´nica de Pediatrı´a, Seccio´n de Neuropediatrı´a, Hospital Universitario Virgen del Rocı´o, Spain Received 17 May 2010; received in revised form 24 September 2010; accepted 27 September 2010
Abstract Rett Syndrome (RS; MIM_312750) is a severe and progressive neurodevelopmental disorder affecting principally females. Mutations in X-Linked MECP2 gene (methyl CpG-binding protein 2; MIM_300005) have been reported as being the major cause of RS. Mutations in this gene have been described as cause of wide spectrum of neurological disorders and mental retardation in males. In some cases, mutations in MECP2 in males produce clinical picture similar to RS. Here we report the identification of the novel truncating mutation Y120X in a 4-year-old child with atypical RS phenotype. Chromosome analysis showed a normal karyotype, and blood DNA and tissue DNA analysis reveal a mosaic for the mutation. Patient’s mother DNA analysis showed that this is a de novo mutation, that has never been described before in any female or male case of RS. Ó 2010 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved. Keywords: Rett Syndrome; MECP2; Somatic mosaicism; Male patient
1. Introduction Rett syndrome (RS) is a X-linked genetic disease and one of the most common causes of genetic mental retardation in females [1]. MECP2 gene (methyl CpGbinding protein (2) was associated to RS due to identification of mutations in this gene in more than 90% of RS females [2] and 1.3–17% in RS males [3,4]. RS is a progressive neurodevelopmental disorder characterized by a
⇑ Corresponding author at: Unidad de Gestio´n Clı´nica de Gene´tica, Reproduccio´n y Medicina Fetal, Hospital Universitario Virgen del Rocı´o, Avenida Manuel Siurot s/n, 41013 Seville, Spain. Tel.: +34 955 012 772. E-mail address: [email protected] (G. Antin˜olo).
normal postnatal development until 6–18 months of age, followed by stagnation and regression of acquired skills and purposeful movement of the hands. Patients develop a severe disorder with secondary microcephaly, central respiratory deregulation, stereotypic hands movements and often have intractable seizures. Initially, MECP2 mutations in males were considered lethal, nevertheless, in the last decade several mutations have been described in males affected with a wide spectrum of neurological disorders, such as severe encephalopathy, Angelman syndrome-like phenotype [5] or X-Linked mental retardation [6]. Furthermore, several male cases with a classical RS phenotype have been described associated to X-Chromosome aneuploidy, or somatic mutation [7]. We report a novel mosaic mutation p.Y120X in a male patient with atypical RS phenotype.
0387-7604/$ - see front matter Ó 2010 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.braindev.2010.09.012
J.I Pieras et al. / Brain & Development 33 (2011) 608–611
2. Patients and methods 2.1. Case report The patient was the first child born from healthy nonconsanguineous parents. At birth, weight was 3900 grs, head circumference was 35 cm and Apgar score was 9 at 1 minute and 10 at 5 minutes. During neonatal period, the child presented global hypotonia and congenital torticollis. At 7 months, he presented normal head circumference, no dysmorphic features and developmental delay with axial hypotonia, mild distal spasticity, reduced global motility and no head control. Visual contact was absent and the patient did not show any interest for environmental stimuli. He had no purposeful use of hands and presented bimanual and oromanual stereotypes, as well as rocking stereotypes. At 2-years-old, the boy presented tonic generalized epileptic seizures, episodes of unmotivated laughing and teeth grinding. Later on, he presented other tonic seizures and partial seizures. At 3-years-old, the patient associated myoclonic jerks provoked by sonorous stimuli, had achieved sedestation, was able to stand up and was starting unstable and ataxic walking with support. He continued with no purposeful use of hands, as well as oromanual and bimanual stereotypes. At this moment, intense visual contact was present, but language was absent and the child presented sleep disorders. Cranial circumference was 50 cm (P50). Cranial MRI, electromyography, electroneurography and screening of metabolic diseases presented normal results. Electroencephalography (EEG) performed when he suffered the first seizures showed epileptic abnormalities with slow spike-wave complex. The clinical picture of continuous manual stereotypes, lack of purposeful use of hands and intense eye contact raised the suspicion of atypical RS. 3. Methods Genomic DNA from the patient and his mother was obtained from blood by standard procedures. In addition, we obtained DNA extracted from patient’s skin sample. An informed consent was obtained from all the participants. The study conformed to the tenets of the declaration of Helsinki and was approved by the “Committee of Ethics and Clinical Investigation” from Hospital Universitario Virgen del Rocı´o. The coding region of MECP2 gene was analyzed by direct sequencing using the Big-Dye Terminator Kit v3.1 and ABI3730 DNA analyzer (Applied Biosystems). Standard peripheral blood cultures were set up. Metaphases were prepared from 72-h culture and 20 G-banded metaphases were analyzed. FISH studies were performed using the protocol provided by the manufacturer (Vysis, Downer, Grove, IL), using chromosome X and Y centromeric probe and evaluating a total of 200 nuclei. To confirm
609
the results chromosomal and FISH analysis were performed on cultured fibroblasts from skin biopsy. MLPA method was employed, using MLPA-P015 kit, purchased from MRC-Holland (Amsterdam, The Netherlands), and according to manufacturer’s recommendations. This MLPA kit contains probes for each of the four exons of MECP2 gene and is designed to detect deletions/duplications. Fragment analyzes were performed using the 3730 DNA analyzer (Applied Biosystems) and for data analysis we used GeneMarker v 1.6 (Softgenetics L.L.C). We normalized the samples by the peak height, and included control individuals who had previously been confirmed to have no CNVs of the studied genes. Exon 3 of the MECP2 gene was PCR amplified from DNA derived from tissue and blood sample, and PCR fragments were subcloned into pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA). 20 clones from each one were then sequenced using the Big-Dye Terminator Kit v3.1 and analyzed on a 3730 sequencer. Finally, we performed a Clustawl analysis, in order to address the interspecies conservation of the methyl-CpG-binding domain (MBD). 4. Results The chromosome analysis from peripheral blood revealed a normal karyotype 46, XY. The same result was observed following karyotype analysis and FISH on cultured fibroblasts from skin biopsy. MLPA studies from blood DNA, revealed a normal gene dosage for MECP2. MECP2 gene sequence analysis revealed the presence of the variant c.360T > G together the wild-type c.360T (NM_015845) in blood DNA. The presence of these 2 variants was confirmed by gene sequencing from DNA obtained from tissue sample (Fig. 1). The analysis of 40 clones with PCR fragments of exon 3 of the MECP2 derived from tissue and blood sample respectively, revealed the presence of the mutated and the normal alleles in different clones, with a ratio of 3/20 and 2/20 (Supplementary Figure). The DNA analyzes of his mother showed no sequence alteration, revealing a de novo origin. The c.360T > G mutation causes a premature stop codon at 120 amino acid (Y120X) within the MBD of MECP2 protein. This region is a specific domain of 72 residues composed of the amino acids between 102–174 positions according to ScanProSite prediction. The Clustawl analysis of these 72 residues, MBD, revealed a highly interspecies conservation. The existence of the variant c.360T > G together with the wild-type c.360T in both blood and tissue samples, and the presence of both variants separated in different clones are indicating somatic mosaicism in our RS patient. Also, the presence of a normal karyotype, and
610
J.I Pieras et al. / Brain & Development 33 (2011) 608–611
Fig. 1. MECP2 sequence electropherograms of blood DNA obtained of the patient (1) tissue DNA obtained of the patient (2) and blood DNA obtained of his mother (3). Mutation c.360 T > G, (p.Y120X), are boxed.
the absence of MECP2 duplications are consistent with this hypothesis. 5. Discussion MECP2 is a silencing gene that controls timing for the inactivation of several functional genes in the perinatal period. Rett Syndrome (RS; MIM_312750) is a severe and progressive neurodevelopmental disorder affecting principally females [8,9]. In the last years, three groups of male patients with mutations in MECP2 gene have been defined. First group is composed of patients who have mutations in MECP2 gene identified previously in classical RS females. Those patients have severe neonatal encephalopathy and usually die within 1–2 years after birth. In addition, It has been identified a subgroup of males with normal life span and typical RS picture. These patients usually present a XXY-karyotype or a somatic mosaicism for the mutation. The second group of male patient’s present mutations not previously described in RS females. These mutations are compatible with adult life and present a wide range of mental retardation. The third group is composed of males having a duplication of the whole MECP2 gene. They present a severe clinical picture, including infantile hypotonia, recurrent respiratory infection, severe mental retarda-
tion, absence of speech development, seizures and spasticity [4]. In this paper, we report the identification of a novel de novo mutation in the MECP2 detected in a 3 yearsold boy. The mutation, namely, Y120X, has not been described before in any female case. The new truncating variation Y120X, is located in the middle of MECP2 functional domain and would produce a truncating protein with an incomplete MBD. In contrast to other RS males with a MECP2-mutation and XXY-karyotype or somatic mosaicism with typical RS picture, that is normally seen in female patients, our patient presents atypical RS phenotype. Although it would be necessary further functional analysis, the alteration of this MBD domain could affect the protein function resulting in atypical RS phenotype in this patient. Thus, RS features and survival of this patient could be explained by the existence of somatic mosaicism.
Acknowledgements We would like to thank the family who participated in the study. The CIBER de Enfermedades Raras is an initiative of the ISCIII. J.I.P. was supported by Fondo de Investigacio´n Sanitaria.
J.I Pieras et al. / Brain & Development 33 (2011) 608–611
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.braindev.2010.09.012. References [1] Ben Zeev Ghidoni B. Rett syndrome. Child Adolesc Psychiatr Clin N Am 2007;16:723–4. [2] Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 1999;23:185–8. [3] Meloni I, Bruttini M, Longo I, Mari F, Rizzolio F, D’Adamo P, et al. A mutation in the rett syndrome gene, MECP2, causes X-linked mental retardation and progressive spasticity in males. Am J Hum Genet 2000;67:982–5.
611
[4] Villard L. MECP2 mutations in males. J Med Genet 2007;44:417–23. [5] Watson P, Black G, Ramsden S, Barrow M, Super M, Kerr B, et al. Angelman syndrome phenotype associated with mutations in MECP2, a gene encoding a methyl CpG binding protein. J Med Genet 2001;38:224–8. [6] Ju¨lich K, Horn D, Burfeind P, Erler T, Auber B. A novel MECP2 mutation in a boy with neonatal encephalopathy and facial dysmorphism. J Pediatr 2009;155:140–3. [7] Topcßu M, Akyerli C, Sayi A, To¨ru¨ner GA, Kocßog˘lu SR, Cimbisß M, et al. Somatic mosaicism for a MECP2 mutation associated with classic Rett syndrome in a boy. Eur J Hum Genet 2002;10:77–81. [8] Matijevic T, Knezevic J, Slavica M, Pavelic J. Rett syndrome: from the gene to the disease. Eur Neurol 2009;61:3–10. [9] Hagberg B, Hanefeld F, Percy A, Skjeldal O. An update on clinically applicable diagnostic criteria in Rett syndrome. Comments to Rett Syndrome Clinical Criteria Consensus Panel Satellite to European Paediatric Neurology Society Meeting, Baden Baden, Germany, 11 September 2001. Eur J Paediatr Neurol 2002;6:293–7.