Chen, Z

Houlden, H

O'Connor, E

Vandrovcova, J

Wood, NW

Yau, WY

English

PubMed

UCL Discovery

GGC Repeat Expansion in NOTCH2NLC is rare in European Patients with Essential Tremor Short running title: The novel GGC expansion is not found in European ET Wai Yan Yau1*, Emer O’Connor1*, Zhongbo Chen1,2, Jana Vandrovcova1, Nicholas W Wood1,3,4, Henry Houlden1,3  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 4 Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, National Hospitial for Neurology and Neurosurgery, London, UK *Both authors contribute equally to this study  Sir, We read with interest that Sun et al. identified GGC repeat expansion in the 5’ region of NOTCH2NLC gene in Chinese patients with essential tremor (ET)1. Recently, two independent research teams have shown that this novel repeat expansion is the causative mutation for neuronal intranuclear inclusion disease (NIID)2, 3. NIID is a progressive neurodegenerative disease with the pathological hallmarks of eosinophilic ubiquitin-positive and p62-positive intranuclear inclusions in both central and peripheral nervous systems, and other tissue organs4-6. Patients can present with either dementia-dominant or weakness-dominant subtypes6; a Parkinson Disease phenocopy has also been recently described7. Tremor is present in approximately one-third of these reported cohorts carrying the GGC repeat expansion3, 8-10, although it invariably manifests with other neurological features such as leukoencephalopathy and cognitive impairment.   In contrast to NIID, ET is a clinical syndrome defined as an isolated tremor syndrome of bilateral upper limb action tremor of at least three years’ duration11. A new addition to the classification is essential tremor plus (ET-plus)11: ET with additional neurological signs of uncertain significance such as impaired tandem gait, ambiguous dystonic posturing or memory impairment. There is no definitive radiological or pathological marker for the ET syndromes; and only a handful of genetic variants are reported in single families and none has been reproducible12. The finding of 11/197 (5.58%) Chinese ET pedigrees carrying the GGC repeat expansion suggests that this mutation may play a significant role in the genetics of ET1. Given all the patients with the GGC repeat expansion in NOTCH2NLC reported in the literatures are East Asians, we are interested to establish the prevalence of this mutation in a European ET cohort.   We analysed 111 index ET patients of European descent who were recruited from the National Hospital for Neurology and Neurosurgery (NHNN). All patients were clinically assessed by neurologists who have interests in neurogenetics and movement disorders. The distinction between ET and ET-plus was made via retrospective review of patients’ clinical records. The study was approved by the joint ethics committee of UCL institute of Neurology and NHNN, UK (UCLH: 04/N034). To test for the presence of GGC repeat expansion at NOTCH2NLC, we carried out repeat primed PCR (RP-PCR) on all patients’ genomic DNA based on published protocol2, followed by fragment length analysis on an ABI 3730xl DNA analyser with a GeneScan 500 LIZ Size Standard (Thermo Fisher Scientific) and GeneMapper software (version 5.0, Scientific)(Sup Table 1)(Sup Fig 1A,C). To avoid false negative results, we repeated the RP-PCR in duplicate with two 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 NOTCH2NLC2 (Sup Fig 1B,D).   In our cohort of 111 index European patients with ET, 35 patients had a family history of autosomal dominant inheritance. “Pure” ET subgroup comprised 74 patients whilst ET-plus subgroup comprised 37 patients. In the latter subgroup, 62% had ambiguous dystonic posturing, 32% had impaired tandem gait, and 5% had equivocal parkinsonism. Table 1 outlines the demographic data of the cohort. In our screening using RP-PCR, we did not identify any patient carrying the GGC repeat expansion in NOTCH2NLC. The repeat sizes of our patients ranged from 9 to 33, with an average of 19.444.02 (Fig 1).   The absence of a positive screening result in our cohort infers that this mutation is unlikely a major contributor to the genetic aetiology of ET in the Europeans. The ethnic difference in mutation prevalence may be a result of a founder effect in East Asian populations. Another example of this is dentatorubral-pallidoluysian atrophy, a CAG repeat expansion disorder, which has a relatively high prevalence in Japan but rare elsewhere13. The GGC repeat sizes of our cohort is similar to those in the Chinese ET patients without expanded GGC repeats. The weaknesses of our study include: (1) our cohort size may not be sufficiently large, especially for familial ET patients; (2) we retrospectively classified patients into the ET and ET-plus subgroups. However, we would still expect to identify patients carrying the GGC repeat expansion in NOTCH2NLC if the prevalence of this mutation is similar in Chinese and European cohorts of ET patients. Further studies with larger cohorts of European patients will help us to better define the role of this mutation for ET, NIID and other movement disorders outside of Japan and China.    Table Table 1. Demographic data of European essential tremor cohort screened.   All Probands ET ET-plus n 111 74 37 Sex, female, n (%) 55 (49.5) 42 (56.8) 13 (35.1) Age at onset, mean (ranges) in years 42.4 (10-70) 45.5 (15-52) 36 (13-70) Tremor              Upper limbs, n (%) 111(100) 74 (100) 37 (100)           Lower limbs, n (%) 12 (10.8) 7 (9.5) 5 (13.5)           Head, n (%) 62 (55.9) 35 (47.3) 27 (73)           Voice, n (%) 21 (18.9) 15 (20.3) 6 (16.2) Ataxia, n (%)* 12 (10.8) 0  12 (32.4) Parkinsonism, n (%)* 2 (1.8) 0 2 (5.4) Dystonia, n (%)* 22 (19.8) 0 22 (59.5) Memory impairment, n (%)* 0 0 0 Family History, n (%) 33 (29.7) 23 (31.1) 10 (27) Figure  Figure 1. Distribution of the GGC repeat length of NOTCH2NLC in the 111 patients with essential tremor from the UK.  Data availability The data supporting the findings of this study are available from the corresponding author upon reasonable request.  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 controls.   Funding W.Y.Y. receives a PhD studentship from Ataxia UK and Rosetree Trust. E.O.C. receives a studentship from the brain research UK. Z.C. is supported by a clinical fellowship from the Leonard Wolfson Foundation. We are grateful to the Medical Research 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.  Competing interests The authors declare no competing interests.  Supplementary materials Supplementary figure 1. (A, C) Representative electropherograms of RP-PCR analysis in a positive control shows a characteristic sawtooth appearance as compared to a patient without the expansion. (B, D) Fragment analysis of a positive control only captured one non-expanded allele (~19 GGC repeats). In contrast, two alleles were captured in a patient in our UK cohort (~15/28 GGC repeats).   Supplementary table 1. Primers sequences and thermocycling conditions                 References 1. Sun QY, Xu Q, Tian Y, et al. Expansion of GGC repeat in the human-specific NOTCH2NLC gene is associated with essential tremor. Brain 2019. 2. 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;51(8):1222-1232. 3. 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;51(8):1215-1221. 4. Liu Y, Mimuro M, Yoshida M, et al. Inclusion-positive cell types in adult-onset intranuclear inclusion body disease: implications for clinical diagnosis. Acta Neuropathol 2008;116(6):615-623. 5. Lindenberg R, Rubinstein LJ, Herman MM, Haydon GB. A light and electron microscopy study of an unusual widespread nuclear inclusion body disease. A possible residuum of an old herpesvirus infection. Acta Neuropathol 1968;10(1):54-73. 6. Sone J, Mori K, Inagaki T, et al. Clinicopathological features of adult-onset neuronal intranuclear inclusion disease. Brain 2016;139(Pt 12):3170-3186. 7. Deng J, Gu M, Miao Y, et al. Long-read sequencing identified repeat expansions in the 5'UTR of the NOTCH2NLC gene from Chinese patients with neuronal intranuclear inclusion disease. J Med Genet 2019. 8. Tian Y, Wang JL, Huang W, et al. Expansion of Human-Specific GGC Repeat in Neuronal Intranuclear Inclusion Disease-Related Disorders. Am J Hum Genet 2019;105(1):166-176. 9. Okubo M, Doi H, Fukai R, et al. GGC Repeat Expansion of NOTCH2NLC in Adult Patients with Leukoencephalopathy. Ann Neurol 2019;86(6):962-968. 10. Deng J, Gu M, Miao Y, et al. Long-read sequencing identified repeat expansions in the 5'UTR of the NOTCH2NLC gene from Chinese patients with neuronal intranuclear inclusion disease. J Med Genet 2019;56(11):758-764. 11. Bhatia KP, Bain P, Bajaj N, et al. Consensus Statement on the classification of tremors. from the task force on tremor of the International Parkinson and Movement Disorder Society. Mov Disord 2018;33(1):75-87. 12. Hopfner F, Helmich RC. The etiology of essential tremor: Genes versus environment. Parkinsonism Relat Disord 2018;46 Suppl 1:S92-S96. 13. Le Ber I, Camuzat A, Castelnovo G, et al. Prevalence of dentatorubral-pallidoluysian atrophy in a large series of white patients with cerebellar ataxia. Arch Neurol 2003;60(8):1097-1099.     

GGC repeat expansion in NOTCH2NLC is rare in European patients with essential tremor

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GGC repeat expansion in NOTCH2NLC is rare in European patients with essential tremor

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