Chen, Z

Houlden, H

Lynch, DS

Sullivan, R

Vandrovcova, J

Wood, NW

Yau, WY

English

UCL Discovery

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/ana.25818  GGC Repeat Expansion in NOTCH2NLC is rare in European Leukoencephalopathy Running head: No novel GGC expansion in European Leukoencephalopathy Wai Yan Yau1* FRACP, Roisin Sullivan1* MSc, Zhongbo Chen1,2 MRCP, David S Lynch1 PhD, Jana Vandrovcova1 PhD, Nicholas W Wood1,3 PhD, Henry Houlden1,3 PhD  1 Department of Neuromuscular Diseases, Institute of Neurology, University College London, UK 2 Department of Neurodegenerative Diseases, Institute of Neurology, University College London, UK 3 Neurogenetics Unit, National Hospital for Neurology and Neurosurgery, London, UK *Both authors contribute equally to this study  Page count: 6 Word count: 383 Number of figure: 1    This article is protected by copyright. All rights reserved.  We read with interest that Okubo et al. identified GGC repeat expansion in NOTCH2NLC as the most frequent genetic cause of adult-onset leukoencephalopathy in Japan1.  After the initial discovery of the causative mutation for neuronal intranuclear inclusion disease (NIID)2, 3, all additional reported NIID patients with this mutation are East Asians. Adult-onset genetic leukodystrophy is clinically and radiologically heterogeneous, making definitive diagnosis challenging. We aim to screen for this novel expansion in a European cohort of adult-onset leukoencephalopathy.   Our cohort consists of 52 adult-onset leukoencephalopathy patients with unknown genetic diagnosis. The patients underwent appropriate investigations to exclude acquired causes, severe small vessel diseases and neuro-metabolic disorders. This was followed by whole-exome sequencing, the protocol of which was previously described4; no pathogenic mutation was identified. They underwent screening using repeat-primed polymerase chain reaction (RP-PCR) following published protocol3 (Fig 1 B, D). To avoid false negative results, we repeated the RP-PCR in triplicate with three different positive controls. In patients without the expansion, we estimated their GGC repeat sizes by amplifying the genomic DNA region containing the repeat using PCR primers specific for NOTCH2NLC3 (Fig 1 C, E). All patients were from the National Hospital for Neurology and Neurosurgery (NHNN) in the United Kingdom (UK) and were clinically assessed by neurologists with interest in leukodystrophy. This article is protected by copyright. All rights reserved.The study was approved by the joint ethics committee of UCL institute of Neurology and NHNN, UK (UCLH: 04/N034).  In our leukoencephalopathy cohort, we did not identify any patient with the GGC repeat expansion in NOTCH2NLC. The estimated repeat sizes ranged from 12 to 26 in our patients (Fig 1A). The main weaknesses of our study include: (1) our modest sample size; (2) single centre recruitment in the UK. Despite of these limitations, our study demonstrates that this novel expansion is likely to be rare in European leukoencephalopathy patients. NIID may have a founder effect in East Asian population, similar to spinocerebellar ataxia type 31, which is a rare form of ataxia with repeat expansion worldwide but a very frequent cause in Japan5.  CSF1R remains the most common genetic cause of adult-onset leukoencephalopathy with European descent4. Further studies are required to establish the exact prevalence of NOTCH2NLC repeat expansion and its role in the aetiology of progressive dementing disorders with white matter changes in the European and other ethnic populations.   Acknowledgement This study is made possible by the generous participation of the patients. We are grateful to Professor Shoji Tsuji from Department of Neurology of the University of Tokyo providing us with genomic DNA of the positive control. W.Y.Y. receives a PhD studentship from Ataxia UK and Rosetree Trust. R.S. receives funding from MRC. Z.C. is supported by a clinical fellowship from the Leonard Wolfson Foundation. We are grateful to the Medical Research This article is protected by copyright. All rights reserved.Council (MRC), The Wellcome Trust Synaptopathies award, MRC Centre grant (G0601943), Ataxia UK, The Rosetrees Trust, Brain Research UK, UCL ODA/LMIC award, The MSA Trust, MDUK, The Muscular Dystrophy Association (MDA). This research was also supported by the UCL/UCLH National Institute for Health Research University College London Hospitals Biomedical Research Centre.    Author contributions WYY, RS, NWW and HH contributed to conception and design of the study; WYY, RS, DSL, JV and ZC contributed to the acquisition and analysis of data; WYY and RS contributed to drafting the text and preparing the figures.  Potential conflicts of interest The authors declare no competing interests.  Data availability The data supporting the findings of this study are available from the corresponding author upon reasonable request. This article is protected by copyright. All rights reserved.         References 1. Okubo M, Doi H, Fukai R, et al. GGC Repeat Expansion of NOTCH2NLC in Adult Patients with Leukoencephalopathy. Ann Neurol. 2019 Dec;86(6):962-8. 2. Sone J, Mitsuhashi S, Fujita A, et al. Long-read sequencing identifies GGC repeat expansions in NOTCH2NLC associated with neuronal intranuclear inclusion disease. Nat Genet. 2019 Aug;51(8):1215-21. 3. Ishiura H, Shibata S, Yoshimura J, et al. Noncoding CGG repeat expansions in neuronal intranuclear inclusion disease, oculopharyngodistal myopathy and an overlapping disease. Nat Genet. 2019 Aug;51(8):1222-32. This article is protected by copyright. All rights reserved.4. Lynch DS, Rodrigues Brandao de Paiva A, Zhang WJ, et al. Clinical and genetic characterization of leukoencephalopathies in adults. Brain. 2017 May 1;140(5):1204-11. 5. Ishikawa K, Durr A, Klopstock T, et al. Pentanucleotide repeats at the spinocerebellar ataxia type 31 (SCA31) locus in Caucasians. Neurology. 2011 Nov 15;77(20):1853-5.          Figure legend Figure 1. (A) Distribution of the GGC repeat length of NOTCH2NLC in the 52 adult-onset leukoencephalopathy patients from the UK. (B, D) Representative electropherogram of RP-PCR analysis in a positive control showed a characteristic sawtooth appearance compared to a patient without the expansion. (C, E) Fragment analysis of a positive control only captured This article is protected by copyright. All rights reserved.one non-expanded allele (~19 GGC repeats). In contrast, two alleles were captured in a patient from our UK cohort (~15/23 GGC repeats).  This article is protected by copyright. All rights reserved.Accepted Article

GGC Repeat Expansion in NOTCH2NLC is rare in European Leukoencephalopathy

https://discovery.ucl.ac.uk/10103159/1/ana.25818.pdf

GGC Repeat Expansion in NOTCH2NLC is rare in European Leukoencephalopathy

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