RESEARCH ARTICLE OFFICIAL JOURNAL Variants Within TSC2 Exons 25 and 31 Are Very Unlikely to Cause Clinically Diagnosable Tuberous Sclerosis ∗ Rosemary Ekong,1 Mark Nellist,2 Marianne Hoogeveen-Westerveld,2 Marjolein Wentink,2 www.hgvs.org Jessica Panzer,3,4 Steven Sparagana,5 Warren Emmett,6 Natalie L. Dawson,7 Marie Claire Malinge,8 Rima Nabbout,9 Caterina Carbonara,10 Marco Barberis,11 Sergio Padovan,12 Marta Futema,13 Vincent Plagnol,6 Steve E. Humphries,13 Nicola Migone,14 and Sue Povey1 1Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, UK; 2Department of Clinical Genetics, Erasmus MC, Rotterdam, 3015CN, The Netherlands; 3Department of Pediatrics, Division of Neurology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-4318; 4Department of Neurology Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; 5Texas Scottish Rite Hospital for Children, Dallas, Texas 75219; 6University College London Genetics Institute, Darwin building, Gower Street, London WC1E 6BT, UK; 7Institute of Structural and Molecular Biology, University College London, London WC1E 6BT, UK; 8UF de Gen´ etique´ Moleculaire,´ Departement´ de Biochimie Gen´ etique´ PBMM, Institut de Biologie en Sante´ CHU Angers, 49933 Angers, Cedex 9, France; 9Centre de Ref´ erence´ des Epilepsies Rares, Hopitalˆ Universitaire Necker - Enfants Malades, 75015 Paris, France; 10Neonatology and Neonatal Intensive Care Unit, S. Anna Hospital, 10126 Torino, Italy; 11Laboratory of Molecular Genetics, Azienda Ospedaliero Universitaria Citta` della Salute e della Scienza, Presidio OIRM S. Anna,10126 Torino, Italy; 12CNR-IBB UOS-TO at MBC, Molecular Biotechnology Center for University of Turin, 10126 Torino, Italy; 13Centre for Cardiovascular Genetics, British Heart Foundation Laboratories, Institute of Cardiovascular Sciences, University College London, London, UK; 14Department of Medical Sciences, University of Turin, 10126 Torino, Italy Communicated by Richard Wooster Received 17 August 2015; accepted revised manuscript 7 December 2015. Published online 25 December 2015 in Wiley Online Library (www.wiley.com/humanmutation). DOI: 10.1002/humu.22951 Hum Mutat 37:364–370, 2016. Published 2015 Wiley Periodi- ABSTRACT: Inactivating mutations in TSC1 and TSC2 ∗ cause tuberous sclerosis complex (TSC). The 2012 inter- cals, Inc. national consensus meeting on TSC diagnosis and manage- KEY WORDS: tuberous sclerosis; diagnosis; TSC2; alterna- ment agreed that the identification of a pathogenic TSC1 tive splicing; variants or TSC2 variant establishes a diagnosis of TSC, even in the absence of clinical signs. However, exons 25 and 31 of TSC2 are subject to alternative splicing. No variants caus- ing clinically diagnosed TSC have been reported in these exons, raising the possibility that such variants would not Introduction cause TSC. We present truncating and in-frame variants in exons 25 and 31 in three individuals unlikely to ful- Tuberous sclerosis complex (TSC) is an autosomal-dominant fil TSC diagnostic criteria and examine the importance of disease caused by inactivating mutations in either TSC1 (MIM these exons in TSC using different approaches. Amino #605284) or TSC2 (MIM #191092). TSC disease severity is variable acid conservation analysis suggests significantly less con- with signs and symptoms ranging from hypomelanotic macules, to servation in these exons compared with the majority of epilepsy, intellectual disability, autism, and multiple hamartomas in TSC2 exons, and TSC2 expression data demonstrates that kidney, brain, heart, and lung. In TSC, about 70% of cases are due to the majority of TSC2 transcripts lack exons 25 and/or 31 new mutations [Sampson et al., 1989; Osborne et al., 1991; Au et al., in many human adult tissues. In vitro assay of both ex- 2007] and this presents a challenge for molecular diagnostics espe- ons shows that neither exon is essential for TSC complex cially when the variant identified is not obviously disease causing. function. Our evidence suggests that variants in TSC2 ex- For example, some missense changes in TSC2 have been associated ons 25 or 31 are very unlikely to cause classical TSC, with TSC in patients diagnosed with definite TSC [Sancak et al., although a role for these exons in tissue/stage specific de- 2005; Hoogeveen-Westerveld et al., 2011], as well as cases in which velopment cannot be excluded. symptoms are less severe and TSC is more often likely to be familial [Khare et al., 2001; O’Connor et al., 2003; Mayer et al., 2004; Jansen et al., 2006; Wentink et al., 2012]. Although there are well-defined clinical diagnostic criteria for Additional Supporting Information may be found in the online version of this article. TSC [Northrup et al., 2013], it can still be difficult to establish a †These authors contributed equally to this work. clinical diagnosis of TSC, particularly in young patients who do not ∗Correspondence to: Rosemary Ekong, Department of Genetics, Evolution & Envi- yet exhibit typical TSC lesions, but may have severe but nonspecific ronment, University College London, Darwin Building, Gower Street, London WC1E 6BT, symptoms such as epilepsy, intellectual disability, and/or autism. In UK. E-mail: [email protected]. these cases, a molecular diagnosis can be helpful. Indeed, the iden- Contract grant sponsors: Tuberous Sclerosis Association (UK); TS Alliance (USA); tification of a clearly inactivating TSC1 or TSC2 mutation is consid- British Heart Foundation (PG08/008); National Institute for Health Research, University ered sufficient evidence for a diagnosis of TSC, even in the absence of College London Hospitals Biomedical Research Centre; Associazione Emma & Ernesto clinical signs [Northrup et al., 2013]. Increasingly, next-generation Rulfo per la Genetica Medica; Wellcome Trust (104960/Z/14/Z, WT091310). sequencing (NGS) of TSC1, TSC2, and many other genes simultane- ∗∗ C 2015 The Authors. Human Mutation published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. ously (gene panels, whole-genome [WGS] or whole-exome [WES] nificance of these variants in TSC, we reviewed all variants mapping sequencing) is being used in diagnostic settings. NGS is also applied to exons 25 and 31 in the TSC2 LOVD (http://www.lovd.nl/TSC2), to population genetics in large-scale sequencing studies, such as the performed conservation analysis, reviewed TSC2 mRNA expres- NHLBI GO Exome Sequencing Project with data in the Exome Vari- sion data, and conducted in vitro functional analysis. We conclude ant Server (EVS) and the Exome Aggregation Consortium (ExAC). that TSC2 exons 25 and 31 are not required for the TSC complex- In these studies, TSC is not assessed and the available phenotype dependent inhibition of TORC1 and that variants in these exons are data are very limited. This reliance on molecular diagnosis makes unlikely to cause TSC. it even more important that the interpretation of any apparently pathological variant is correct. Materials and Methods The main recognized challenge to the molecular diagnosis of TSC has been (and still is) the “Variants of Uncertain Significance” TSC2 Exon and Variant Nomenclature (VUS), many of these being missense and in-frame indels. The TSC1 and TSC2 genes encode core components of the TSC protein TSC2 (GenBank NG 005895.1 GI:125662814) consists of 42 complex that is a critical negative regulator of the mechanistic tar- exons, including a 5’ noncoding exon [ftp://ftp.ebi.edu.au/pub/ get of rapamycin (mTOR) complex 1 (TORC1) [Dibble and Man- databases/lrgex/LRG_487.xml]. However, the TSC community usu- ning, 2013]. In vitro assays to determine the effects of TSC1 and ally number TSC2 exons from the start of the protein coding se- TSC2 missense and in-frame indels on TORC1 activity have proven quence making 41 exons in total, with the 5’ noncoding exon num- useful in the ascertainment of the pathogenicity of variants previ- bered 1a. This convention is used here. ously reported as VUS [Hoogeveen-Westerveld et al., 2011, 2013; Nucleotide numbering corresponds to the TSC2 cDNA sequence Dunlop et al., 2011]. In these tests, assay results are summarized (GenBank NM 000548.3, GI:116256351) with +1asAoftheATG as the effect of the variants on the “TSC complex function” by translation initiation codon in the reference sequence, and the ini- which we mean “the inhibition of TORC1 activity.” An additional tiation codon as codon 1. option for evaluating a VUS is to perform conservation analysis of orthologous protein sequences in multiple species. Several com- Patients and Variants putational algorithms (e.g., PolyPhen, SIFT) incorporate protein alignments from multiple species into the evaluation of the effects Case 1: A predicted nonsense variant in TSC2 exon 25 (c.2859dup, of amino acid substitutions on protein function [Ng and Henikoff, p.K954Qfs∗6) was identified in an epilepsy gene panel test performed 2003; Adzhubei et al., 2013]. In our experience, these algorithms on DNA from a 10-year-old patient with epilepsy, but no clinical or were not reliable enough to classify individual VUS with confidence radiographic findings supporting a diagnosis of TSC. The variant [Hoogeveen-Westerveld et al., 2011]. However, this approach may was reported as “pathogenic.” still be applicable where high-quality alignments of multiple species Case 2: We analyzed