Characterization of a Novel Transcript of the EHMT1 Gene Reveals Important Diagnostic Implications for Kleefstra Syndrome
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Characterization of a novel transcript of the EHMT1 gene reveals important diagnostic implications for Kleefstra Syndrome Willy M Nillesen, Helger G Yntema, Marco Moscarda, Nienke E Verbeek, Louise C. Wilson, Frances Cowan, Marga Schepens, Annick Raas-Rothschild, Orly Orly Yehouda -Weinstein, Marcella Zollino, et al. To cite this version: Willy M Nillesen, Helger G Yntema, Marco Moscarda, Nienke E Verbeek, Louise C. Wilson, et al.. Characterization of a novel transcript of the EHMT1 gene reveals important diagnostic implications for Kleefstra Syndrome. Human Mutation, Wiley, 2011, 32 (7), pp.853. 10.1002/humu.21523. hal- 00655181 HAL Id: hal-00655181 https://hal.archives-ouvertes.fr/hal-00655181 Submitted on 27 Dec 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Human Mutation Characterization of a novel transcript of the EHMT1 gene reveals important diagnostic implications for Kleefstra Syndrome For Peer Review Journal: Human Mutation Manuscript ID: humu-2010-0547.R2 Wiley - Manuscript type: Research Article Date Submitted by the 07-Apr-2011 Author: Complete List of Authors: Nillesen, Willy; Radboud University Nijmegen Medical Centre, 849 Department of Human Genetics Yntema, Helger; Radboud University Nijmegen Medical Centre, 849 Department of Human Genetics Moscarda, Marco; Catholic University, Rome, Italy, Institute of Medical Genetics Verbeek, Nienke; UMC Utrecht, Utrecht, The Netherlands, 3Department of Medical Genetics Wilson, Louise; Great Ormond Street Hospital for Children NHS Trust and Institute of Child Health, University College London, UK, Clinical Genetics Unit Cowan, Frances; Imperial College Healthcare Trust, London, UK, Perinatal Neurology Department of Paediatrics Schepens, Marga; Radboud University Nijmegen Medical Centre, 849 Department of Human Genetics Raas-Rothschild, Annick; hadassah hebrew University Medical Center Orly Yehouda –Weinstein, Orly; hadassah hebrew University Medical Center Zollino, Marcella; Catholic University, Rome, Italy, Institute of Medical Genetics vijzelaar, R; 7MRC Holland bv, Amsterdam, the Netherlands Neri, Giovanni; Catholic University, Rome, Italy, Institute of Medical Genetics Nelen, Marcel; Radboud University Nijmegen Medical Centre, 849 Department of Human Genetics; UMC Utrecht, Utrecht, The Netherlands, 3Department of Medical Genetics van Bokhoven, Hans; Radboud University Nijmegen Medical Centre, 849 Department of Human Genetics Giltay, Jacques; University Medical Center, Utrecht, The Netherlands, Department of Biomedical Genetics Kleefstra, Tjitske; Radboud University Nijmegen Medical Centre, 849 Department of Human Genetics John Wiley & Sons, Inc. Page 1 of 29 Human Mutation 1 2 3 Kleefstra syndrome , Chromosome 9q34 microdeletion, 9qSTDS, 4 Key Words: 5 EHMT1 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 2 of 29 1 2 Characterization of a novel transcript of the EHMT1 gene reveals 3 4 5 important diagnostic implications for Kleefstra Syndrome 6 7 8 1† 1† 2 3 9 Willy M Nillesen , Helger G Yntema , Marco Moscarda , Nienke E. Verbeek , 10 4 5 1 6 11 Louise C Wilson , Frances Cowan , Marga Schepens , Annick Raas-Rothschild , 12 6 2 7 2 13 Orly Gafni-Weinstein , Marcella Zollino , Raymon Vijzelaar , Giovanni Neri , Marcel 14 1,3 1 3 1 15 Nelen , Hans van Bokhoven , Jacques Giltay , Tjitske Kleefstra 16 17 1 Department of Human Genetics, RUNMC, Nijmegen, The Netherlands 18 2 Institute of Medical Genetics, Catholic University, Rome, Italy 19 3Department of Medical Genetics, UMC Utrecht, Utrecht, The Netherlands 4 20 Clinical Genetics Unit, Great OrmondFor Street PeerHospital for Children Review NHS Trust and Institute of Child Health, University College London, UK 21 5 Perinatal Neurology, Department of Paediatrics, Imperial College Healthcare Trust, London, UK 22 6 Department of Human Genetics and Metabolic Diseases, Hadassah Hebrew University Medical 23 Center, Jerusalem, Israel 24 7MRC Holland bv, Amsterdam, the Netherlands 25 † 26 These authors contributed equally to this manuscript 27 28 29 30 Corresponding author: 31 Tjitske Kleefstra MD, PhD 32 33 849 Department of Human Genetics 34 35 PO Box 9101 36 37 6500 HB Nijmegen 38 39 The Netherlands 40 41 0031-(0)243613946 42 43 44 45 46 47 Key words: Kleefstra syndrome , Chromosome 9q, Subtelomere deletion, 9qSTDS, 48 49 EHMT1 50 51 52 53 54 1 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 3 of 29 Human Mutation 1 2 ABSTRACT 3 4 5 The core phenotype of Kleefstra syndrome (KS) is characterized by intellectual 6 7 disability, childhood hypotonia and a characteristic facial appearance. This can be 8 9 caused by either submicroscopic 9q34 deletions or loss of function mutations of the 10 11 EHMT1 gene. Remarkably, in three patients with a clinical suspicion of KS, molecular 12 13 cytogenetic analysis revealed an interstitial 9q34 microdeletion proximal to the coding 14 15 region of the EHMT1 gene based on the NM_024757.3 transcript. Since we found a 16 17 mono-allelic EHMT1 transcript suggestive for haploinsufficiency of EHMT1 in two of 18 19 these patients tested, we hypothesized that a deletion of regulatory elements or so 20 For Peer Review 21 far unknown coding sequences in the 5’ region of the EHMT1 gene, might result in a 22 23 phenotype compatible with KS. We further characterized the molecular content of 24 25 deletions proximal to the transcript NM_024757.3 and confirmed presence of a novel 26 Deleted: This new EHMT1 transcript 27 predicted open reading frame comprising 27 coding exons (NM_024757.4). Further therefore. 28 29 analysis showed that all three deletions included the presumed novel first exon of the 30 31 EHMT1 gene and subsequent testing of 75 individuals without previously detectable 32 33 EHMT1 aberrations, showed one additional case with a deletion comprising only this 34 35 5’ part of the gene. 36 37 These results have important implications for the genetic screening of KS and for 38 39 studies of the functional significance of EHMT1 . 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 2 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 4 of 29 1 2 INTRODUCTION 3 4 5 6 Kleefstra syndrome (MIM# 610253) also known as 9q Subtelomeric Deletion 7 8 Syndrome; 9qSTDS, is characterized by intellectual disability, childhood hypotonia, a 9 10 characteristic facial appearance and in some patients, by congenital heart and renal 11 defects, (brachy)microcephaly, epilepsy, obesity and behavioural problems as well 12 13 (Stewart et al., 2004; Yatsenko et al., 2005; Stewart and Kleefstra, 2007; Kleefstra et 14 15 al., 2005, 2006, 2009; Verhoeven et al., 2010). The syndrome can be caused by 16 17 either submicroscopic deletions of the distal long arm of chromosome 9q or 18 19 intragenic loss of function mutations in the EHMT1 (Euchromatic Histone 20 For Peer Review 21 Methyltransferase 1 ) gene (MIM# 607001), both leading to haploinsufficiency of the 22 23 EHMT1 gene. Since the identification of this gene, we have provided regular 24 25 diagnostic services for mutational analysis comprising direct sequencing of the 26 27 coding region and screening for deletions by Multiplex Ligation-dependent Probe 28 29 Amplification (MLPA) of the transcript NM_024757.3 (http://www.ncbi.nlm.nih.gov/). 30 31 So far, we have published information about 27 cases with EHMT1 deletions or 32 33 mutations (Kleefstra et al., 2006, 2009). 34 35 Remarkably, in three patients with a clinical suspicion of Kleefstra syndrome (KS), 36 37 molecular cytogenetic analysis revealed a submicroscopic interstitial 9q deletion 38 39 proximal to the coding region of the EHMT1 gene. We therefore hypothesized that 40 41 regulatory elements or so far unknown coding sequences in the 5’ region of the 42 43 EHMT1 gene, might be affected by the deletion resulting in a phenotype compatible 44 45 with KS. In this study we aimed to further characterize the molecular content of 46 47 deletions proximal to the transcript NM_024757.3 causing KS and test a large group 48 49 of patients suspected of having KS with this hitherto negative findings. 50 51 52 53 54 3 55 56 57 58 59 60 John Wiley & Sons, Inc. Page 5 of 29 Human Mutation 1 2 PATIENTS AND METHODS 3 4 5 6 Patients 7 8 9 All patients were referred to our centre for further genetic analysis because of the 10 clinical suspicion of Kleefstra syndrome. Informed consent was obtained for further 11 12 publication of data and photographs. 13 14 15 16 Patient 1 (Figure 1A,B) was born by vaginal delivery at 40 weeks gestation after a 17 18 pregnancy complicated by first trimester bleedings, treated with progesterone, and 19 20 contractions at 17 weeks, treatedFor with Peerisoxsuprine. Her Review birth weight was 3020 g (25- 21 22 50 th centile), birth length 47 cm (10-25 th centile) and head circumference 32 cm (10- 23 24 25 th centile). Her APGAR scores were 8/9 at 1/5 minutes. During the first day after 25 26 birth, she was bradycardic and generally hypotonic. She suffered from gastro- 27 28 esophageal reflux until 8 months of age. Reduced head circumference centile was 29 30 noted at 5 months. Sleep, feeding and bowel transit and an EEG and 31 32 echocardiography were normal.