European Journal of Human Genetics (2011) 19, 102–107 & 2011 Macmillan Publishers Limited All rights reserved 1018-4813/11 www.nature.com/ejhg ARTICLE Duplications of FOXG1 in 14q12 are associated with developmental epilepsy, mental retardation, and severe speech impairment Nicola Brunetti-Pierri*,1,14, Alex R Paciorkowski2, Roberto Ciccone3, Erika Della Mina3, Maria Clara Bonaglia4, Renato Borgatti4, Christian P Schaaf1, V Reid Sutton1, Zhilian Xia1, Naftha Jelluma5, Claudia Ruivenkamp6, Mary Bertrand2, Thomy JL de Ravel7, Parul Jayakar8, Serena Belli9, Katia Rocchetti10, Chiara Pantaleoni11, Stefano D’Arrigo11, Jeff Hughes1, Sau Wai Cheung1, Orsetta Zuffardi3,12 and Pawel Stankiewicz1,13 Genome-wide high-resolution array analysis is rapidly becoming a reliable method of diagnostic investigation in individuals with mental retardation and congenital anomalies, leading to the identification of several novel microdeletion and microduplication syndromes. We have identified seven individuals with duplication on chromosome 14q11.2q13.1, who exhibited idiopathic developmental delay and cognitive impairment, severe speech delay, and developmental epilepsy. Among these cases, the minimal common duplicated region on chromosome 14q11.2q13.1 includes only three genes, FOXG1, C14orf23, and PRKD1. We propose that increased dosage of Forkhead Box G1 (FOXG1) is the best candidate to explain the abnormal neurodevelopmental phenotypes observed in our patients. Deletions and inactivating mutations of FOXG1 have been associated with a Rett-like syndrome characterized by hypotonia, irritability, developmental delay, hand stereotypies, and deceleration of head growth. FOXG1, encoding a brain-specific transcription factor, has an important role in the developing brain. In fact, in vivo studies in chicken brain demonstrated that overexpression of FOXG1 results in thickening of the neuroepithelium and outgrowth of the telencephalon and mesencephalum, secondary to a reduction in neuroepithelial cell apoptosis. European Journal of Human Genetics (2011) 19, 102–107; doi:10.1038/ejhg.2010.142; published online 25 August 2010 Keywords: FOXG1; developmental delay; speech delay; infantile spasms; array CGH INTRODUCTION phenotypic overlap between the neurological abnormalities seen in Deletions and inactivating mutations of the Forkhead Box G1 (FOXG1) patients with increases or decreases in MECP2 dosage.9,10 To date, gene on chromosome 14q12 have been associated with a Rett-like duplication of the chromosomal region encompassing FOXG1 has neurodevelopmental disorder characterized by developmental delay, been reported in only one patient with infantile spasms, severe hypotonia, irritability, repetitive hand stereotypies, and deceleration of intellectual impairment, and minor dysmorphisms.11 head growth (OMIM 613454).1–7 The phenotypic similarities between Here, we present the clinical and molecular findings in six patients classic Rett syndrome (OMIM 312750), which results from mutations with duplications of the 14q12 region containing the FOXG1 gene. A or deletions in the methyl-CpG binding protein 2 (MECP2)gene,and query of the Database of Chromosomal Imbalance and Phenotype in the neurodevelopmental disorder caused by mutations or deletions in Humans using Ensembl Resources (DECIPHER; https://decipher. FOXG1 suggest that the disruption of one or more common neuronal sanger.ac.uk)12 revealed a seventh patient (DECIPHER 248559) developmental pathway(s) is responsible for these conditions. None- presenting with developmental delay and cognitive impairment and theless, important clinical differences exist between the two syn- a 14q12 duplication involving the FOXG1 gene. dromes. For example, patients with FOXG1 mutations/deletions tend to exhibit abnormal development in early infancy, while also lacking the characteristic autonomic disturbances of Rett syndrome. MATERIALS AND METHODS In male patients, duplications of MECP2 result in delayed DNA samples milestones, infantile hypotonia, progressive spasticity, severe mental Patients were referred for clinical array-CGH analysis to either Medical Genetics disability, absence of language, and increased susceptibility to recur- Laboratories at Baylor College of Medicine (BCM) in Houston, TX, USA (cases rent infections (OMIM 300260).8 Interestingly, there is a significant 1–3) or to the Cytogenetic Laboratory of the University of Pavia in Pavia, Italy 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; 2Division of Pediatric & Developmental Neurology, Department of Neurology, Washington University in St Louis, St Louis, MO, USA; 3Biologia Generale e Genetica Medica, Universita` di Pavia, Pavia, Italy; 4Scientific Institute Eugenio Medea, Bosisio Parini, Lecco, Italy; 5Ipse de Bruggen, Centre for People with Intellectual Disability, Zwammerdam, The Netherlands; 6Department of Clinical Genetics, Center for Human and Clinical Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands; 7Centre for Human Genetics, Leuven, Belgium; 8Division of Genetics and Metabolism, Miami Children’s Hospital, Miami, FL, USA; 9Servizio di Genetica Medica Dip. Laboratorio APSS, Trento, Italy; 10NPI Ospedale Santa Chiara, Trento, Italy; 11Developmental Neurology Unit, C Besta Foundation Neurological Institute, Milan, Italy; 12IRCCS C. Mondino, Pavia, Italy; 13Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland *Correspondence: Dr N Brunetti-Pierri, Telethon Institute of Genetics and Medicine, Via P Castellino, 111, 80131 Napoli, Italy. Tel: +39 081 6132361; Fax: +39 081 5609877; E-mail: [email protected] 14Present address: Department of Pediatrics, Federico II University of Naples, Italy and Telethon Institute of Genetics and Medicine, Naples, Italy. Received 21 January 2010; revised 6 July 2010; accepted 16 July 2010; published online 25 August 2010 FOXG1 duplications N Brunetti-Pierri et al 103 (cases 4–6). Case 7 was obtained through a query of the DECIPHER database Long-range PCR and DNA sequencing and is referred to as DECIPHER 248559. Long-range PCR used to amplify the predicted junction fragments from the Once informed consents, approved by the Institutional Review Board for breakpoint regions in patient 3, which was performed as per the manufacturer’s Human Subject Research at the corresponding institutions, were obtained, instructions (Takara Bio Inc., Otsu, Japan). PCR products were purified with the DNA samples were collected from the probands and their family members. PCR Purification Kit (Qiagen, Valencia, CA, USA) and bidirectionally sequenced DNA was extracted from whole blood using the Puregene DNA extraction kit using the Sanger’s dideoxynucleotide method (Lone Star, Houston, TX, USA). (Gentra, Minneapolis, MN, USA) according to the manufacturer’s instructions. Bioinformatics and in silico sequence analysis Array CGH Genomic sequences based on the oligonucleotide coordinates from the array The 14q duplications in cases 1–3 were identified by screening the Medical CGH experiment were downloaded from the UCSC genome browser (Build 36, Genetics Laboratories at BCM database of array CGH results for over 11 000 UCSC genome browser, March 2006). Interspersed repeat sequences were patients tested using oligonucleotide-based Chromosomal Microarray Analysis analyzed by RepeatMasker (http://www.repeatmasker.org). Regional assemblies (CMA Versions 6, 7, and 8 OLIGO, Baylor College of Medicine, Houston, TX, were done using NCBI BLAST 2 and the Sequencher software (Gene Codes, USA).13,14 Approximately 4700 of these patients were tested using the v6 Ann Arbor, MI, USA). OLIGO (44K array), 5145 patients using the v7 OLIGO (105K array), and 1800 using the v8 OLIGO (180K array). The rearrangements in patients 2 and 3 were PATIENT REPORTS further investigated using a high-resolution 244K Agilent Technologies (Santa Patient 1 Clara, CA, USA) and 2.1 M Roche NimbleGen arrays (Madison, WI, USA), This 4-year-old boy was initially evaluated for a history of infantile spasms and respectively. Cases 4-6 were identified by the cytogenetics laboratory at the global developmental delay. He was born with unilateral postaxial polydactyly, University of Pavia, from a search of the 3752 cases analyzed. Patient 5 was which was corrected surgically, and he underwent strabismus repair at 2 years evaluated using array-CGH with a 44K array (Agilent Technologies). Patients 4 of age. At the age of 6 months, he developed infantile spasms. The electro- and 6 were first examined with the same 44K platform before running them on encephalogram (EEG) performed at that time documented hypsarrhythmia a 180K array (Agilent). All experiments were performed according to the with electrodecrement. Therapy was initiated with adrenocorticotropic hor- 15 manufacturer’s instructions with some modifications. The slides were mone, and a repeat EEG performed 1 month later showed improvement in the scanned into image files using a GenePix Model 4000B microarray scanner background rhythm, although there was a persistence of both right lateral sharp (Molecular Devices, Sunnyvale, CA, USA) or an Agilent G2565 laser scanner. waves and right lateral diffuse slowing. He has been seizure-free on lamotrigine Microarray image files of oligo arrays were quantified using Nimblescan v2.5 for 3 years and his most recent EEG at the age of 4 years showed an 8 Hz (Roche NimbleGen) or Feature Extraction version 9.0 (Agilent Technologies), posterior dominant rhythm. and text file outputs from the quantitation analysis were