Trinucleotide repeat disorders are a heterogeneous group of diseases caused by the expansion, beyond a pathogenic threshold, of unstable DNA tracts in different genes. Sequence interruptions in the repeats have been described in the majority of these disorders and may influence disease phenotype and heritability. Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by a CAG trinucleotide expansion in the androgen receptor ( AR) gene. Diagnostic testing and previous research have relied on fragment analysis polymerase chain reaction to determine the AR CAG repeat size, and have therefore not been able to assess the presence of interruptions. We here report a sequencing study of the AR CAG repeat in a cohort of SBMA patients and control subjects in the United Kingdom. We found no repeat interruptions to be present, and we describe differences between sequencing and traditional sizing methods. © 2014 The Authors

Collins, T

Devoy, A

Fisher, EMC

Fratta, P

Giunti, P

Hanna, MG

Nethisinghe, S

Pemble, S

Sweeney, MG

UCL Discovery

lable at ScienceDirectNeurobiology of Aging 35 (2014) 443.e1e443.e3Contents lists avaiNeurobiology of Agingjournal homepage: www.elsevier .com/locate/neuagingNegative resultsSequencing analysis of the spinal bulbar muscular atrophy CAG expansion revealsabsence of repeat interruptionsqPietro Fratta a,c,1,*, Toby Collins a,1, Sally Pemble d, Suran Nethisinghe b, Anny Devoy a, Paola Giunti b,d,Mary G. Sweeney d, Michael G. Hanna c, Elizabeth M.C. Fisher a,caDepartment of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, UKbDepartment of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, UKcMRC Centre for Neuromuscular Disease, UCL Institute of Neurology, Queen Square, London, UKdNeurogenetics Unit, National Hospital for Neurology and Neurosurgery, Queen Square, London, UKa r t i c l e i n f oArticle history:Received 9 July 2013Accepted 19 July 2013Available online 13 September 2013Keywords:Kennedy’s diseaseSpinal bulbar muscular atrophyCAGTrinucleotideInterruptionsq This is an open-access article distributed undCommons Attribution-NonCommercial-ShareAlike Licommercial use, distribution, and reproduction inoriginal author and source are credited.* Corresponding author at: Department of Neurodetute of Neurology, Queen Square, London WC1N 3BG, Ufax: þ44 (0)2078378047.E-mail address: p.fratta@prion.ucl.ac.uk (P. Fratta).1 Both Authors contributed equally to the work.0197-4580/$ e see front matter  2014 The Authors.http://dx.doi.org/10.1016/j.neurobiolaging.2013.07.015a b s t r a c tTrinucleotide repeat disorders are a heterogeneous group of diseases caused by the expansion, beyond apathogenic threshold, of unstable DNA tracts in different genes. Sequence interruptions in the repeatshave been described in the majority of these disorders and may inﬂuence disease phenotype and her-itability. Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by a CAG trinucleotideexpansion in the androgen receptor (AR) gene. Diagnostic testing and previous research have relied onfragment analysis polymerase chain reaction to determine the AR CAG repeat size, and have therefore notbeen able to assess the presence of interruptions. We here report a sequencing study of the AR CAGrepeat in a cohort of SBMA patients and control subjects in the United Kingdom. We found no repeatinterruptions to be present, and we describe differences between sequencing and traditional sizingmethods. 2014 The Authors. Published by Elsevier Inc. All rights reserved.1. Introduction Interruptions have been shown to play a role in determiningSpinal bulbar muscular atrophy (SBMA), also known as Ken-nedy’s disease, is a slowly progressive, adult-onset, motor neurondisease, inherited in an X-linked recessive manner and caused by aCAG repeat expansion in the ﬁrst exon of the androgen receptorgene (AR) (La Spada et al., 1991). Although normal individuals carry9 to 36 CAG repeats, SBMA patients carry 38 to 62 repeats (La Spadaet al., 1991). SBMA is part of a wider group of neurological diseasesinwhich the causative genetic defect is a coding CAG expansion andfor which the pathogenic threshold is approximately 35 to 40 re-peats (Table 1) (Atsuta et al., 2006; Orr and Zoghbi, 2007). Inter-estingly, although most frequently the CAG expansions are pureruns of CAGs, sequence interruptions have been documented in themajority of these disease genes (Table 1).er the terms of the Creativecense, which permits non-any medium, provided thegenerative Diseas, UCL Insti-K. Tel.: þ44 (0)2034484448;Published by Elsevier Inc. All righthe clinical phenotype and the disease heritability, and thereforecarry potentially important information for genetic testing andcounseling (Chong et al., 1995; Matsuyama et al., 1999; Yu et al.,2011). Pulic genomic databases report rare sequence variations(rs62636527, rs62636528, rs62636529) occurring with low fre-quency (0.013e0.043) in the AR CAG repeat, but SBMA diagnostictesting and previous studies have mostly used fragment sizinganalysis to assess the presence and size of CAG repeats (Atsuta et al.,2006; La Spada et al., 1991), and have not been able to systemati-cally address whether there are interruptions in SBMA expansions,thus warranting direct target sequence analysis.2. MethodsDetails of the patient cohorts, fragment sizing, and sequencingare given in the Supplementary text. The studywas approved by thelocal ethical committee.3. ResultsTo investigate the presence of interruptions in the AR CAGrepeat, we sequenced the AR CAG repeat in a population of 40 SBMApatients and 93 control subjects in the United Kingdom. Overall,SBMA samples showed a median of 45 repeats (range, 40e53ts reserved.Table 1Summary of CAG characteristics in diseases caused by CAG coding expansionsDisease Gene No. of CAG repeats Presence ofinterruptions inCAG repeatsequenceReferenceNormalalleleIntermediatealleleReduced penetrancealleleFully penetrantalleleDRPLA ATN1 6e35 36e47 48e93 No Koide et al., 1994SCA1 ATXN1 6e38 36e38 39e91 Yes Orr et al., 1993SCA2 ATXN2 14e31 32e270 Yes Pulst et al., 1996SCA3 ATXN3 12e44 45e51 52e86 Yes Kawaguchi et al., 1994SCA17 TBP 25e40 41e48 49e66 Yes Koide et al., 1999HD HTT 26 27e35 36e39 40 Yes Huntington’s Disease Collaborative Research Group, 1993SBMA AR 9e36 37 38e62 No La Spada et al., 1991; present workKey: SBMA, spinal bulbar muscular atrophy.P. Fratta et al. / Neurobiology of Aging 35 (2014) 443.e1e443.e3443.e2repeats; Supplementary Fig. 1), which is comparable to the resultsreported in previous U.S. and Japanese series (Atsuta et al., 2006; LaSpada et al., 1991). The quality of our direct sequencing of the CAGexpansion allowed us to address the presence of interruptions byvisual inspection in both forward and reverse sequences of theamplicon (Supplementary Fig. 2). We did not ﬁnd interruptions inany of our SBMA case patients or control subjects (Table 1).Interestingly, when comparing sizing obtained by sequencingto the fragment analysis used diagnostically, we observed aconstant difference of 3 to 4 repeats, with estimation fromfragment analysis being smaller. This was not dependent on theallele size, and was constant among pathological alleles andcontrols.4. DiscussionDiagnostic testing and previous large-scale studies have reliedon polymerase chain reaction fragment analysis for sizing the CAGrepeat in AR, and have therefore not been able to address thepresence of sequence interruptions in a large number of samples(Atsuta et al., 2006; La Spada et al., 1991). This is an importantissue, because interruptions have been shown to be present in themajority of CAG expansion diseases and to play a role in clinicalfeatures of the disease, such as age of onset and presentation.In the case of ataxin 2 (ATXN2), the combination of presence ofinterruptions and repeat size determines very distinct clinicalpresentations that encompass SCA2, L-DOPAeresponsive parkin-sonism, and amyotrophic lateral sclerosis (Matsuyama et al., 1999;Yu et al., 2011). Interruptions have also been shown to inﬂuencethe capability of the repeat to expand in the germ line, thereforecarrying potential implications for genetic counseling (Chunget al., 1993).Importantly, our results show that interruptions are notfrequent in the AR CAG repeat and are therefore not likely to play arole in disease, making SBMA an exception among coding CAGexpansion diseases.Our results also show that fragment analysis, used routinely forthe diagnostic testing of the SBMA and other CAG expansion dis-eases, underestimates the CAG expansion. This discrepancy is pre-sent also when analysing CAG expansions from other genes (Menonet al., 2013). In diagnostic laboratories, this bias is corrected for bycalibrating the fragment analysis with amplicons of known sizes.Our results indicate that direct sequencing of the AR CAG repeatpolymerase chain reaction amplicon in males is a reliable methodfor sizing evaluation, and therefore we suggest that this may beuseful when analyzing size lengths of the CAG repeat near the dis-ease threshold of 38 repeats. This possibility is unique to SBMAtesting in males and differs from all other triplet expansion diseasesin which the presence of sequence variability and the effect ofhaving 2 alleles, makes direct polymerase chain reaction sequencingless informative.Disclosure statementThe authors declare no actual of potential conﬂicts of interest.AcknowledgementsThis work was supported by Medical Research Council/MotorNeurone Disease Association Lady Edith Wolfson Fellowship (P.F.),and by grants from the Medical Research Council, the Motor Neu-rone Disease Association, and the Thierry Latran Foundation (E.F.).Appendix A. Supplementary dataSupplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging.2013.07.015.ReferencesAtsuta, N., Watanabe, H., Ito, M., Banno, H., Suzuki, K., Katsuno, M., Tanaka, F.,Tamakoshi, A., Sobue, G., 2006. Natural history of spinal and bulbarmuscular atrophy (SBMA): a study of 223 Japanese patients. Brain 129,1446e1455.Chong, S.S., McCall, A.E., Cota, J., Subramony, S.H., Orr, H.T., Hughes, M.R.,Zoghbi, H.Y., 1995. Gametic and somatic tissueespeciﬁc heterogeneity of theexpanded SCA1 CAG repeat in spinocerebellar ataxia type 1. Nature Genetics10, 344e350.Chung, M.Y., Ranum, L.P., Duvick, L.A., Servadio, A., Zoghbi, H.Y., Orr, H.T., 1993.Evidence for a mechanism predisposing to intergenerational CAGrepeat instability in spinocerebellar ataxia type I. Nat. Genet. 5,254e258.Huntington’s Disease Collaborative Research Group, 1993. A novel gene containing atrinucleotide repeat that is expanded and unstable on Huntington’s diseasechromosomes. Cell 72, 971e983.Kawaguchi, Y., Okamoto, T., Taniwaki, M., Aizawa, M., Inoue, M., Katayama, S.,Kawakami, H., Nakamura, S., Nishimura, M., Akiguchi, I., Kimura, J., Narumiya, S.,Kakizuka, A., 1994. CAG expansions in a novel gene for Machado-Joseph diseaseat chromosome 14q32.1. 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Sequencing analysis of the spinal bulbar muscular atrophy CAG expansion reveals absence of repeat interruptions

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Sequencing analysis of the spinal bulbar muscular atrophy CAG expansion reveals absence of repeat interruptions

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