Genetic and functional analysis of TBK1 variants in Korean patients with sporadic amyotrophic lateral sclerosis

Accepted Manuscript Genetic and Functional Analysis of TBK1 Variants in Korean Patients with Sporadic Amyotrophic Lateral Sclerosis Young-Eun Kim, Ki-Wook Oh, Min-Young Noh, Minyeop Nahm, Jinseok Park, Su Min Lim, Ja-Hyun Jang, Eun-Hae Cho, Chang-Seok Ki, Seungbok Lee, Seung Hyun Kim PII:

S0197-4580(16)30290-1

DOI:

10.1016/j.neurobiolaging.2016.11.003

Reference:

NBA 9769

To appear in:

Neurobiology of Aging

Received Date: 6 July 2016 Revised Date:

11 October 2016

Accepted Date: 11 November 2016

Please cite this article as: Kim, Y.-E., Oh, K.-W., Noh, M.-Y., Nahm, M., Park, J., Lim, S.M., Jang, J.-H., Cho, E.-H., Ki, C.-S., Lee, S., Kim, S.H., Genetic and Functional Analysis of TBK1 Variants in Korean Patients with Sporadic Amyotrophic Lateral Sclerosis, Neurobiology of Aging (2016), doi: 10.1016/ j.neurobiolaging.2016.11.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Genetic and Functional Analysis of TBK1 Variants in Korean Patients with Sporadic Amyotrophic Lateral Sclerosis

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Young-Eun Kima,1, Ki-Wook Ohb,c,1, Min-Young Nohb,c,1, Minyeop Nahmc, Jinseok Parkb,c, Su Min Limc, Ja-Hyun Janga, Eun-Hae Choa, Chang-Seok Kid,*, Seungbok Leee, Seung Hyun

a

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Kimb,c,**

Green Cross Genome, Yongin, Republic of Korea

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Department of Neurology, Hanyang University College of Medicine, Seoul, Republic of

Korea

Cell Therapy Center, Hanyang University Hospital, Seoul, Republic of Korea

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Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan

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University School of Medicine, Seoul, Republic of Korea e

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Department of Brain and Cognitive Sciences, College of Natural Sciences, Seoul National

University, Seoul, Republic of Korea

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Corresponding author at: Department of Laboratory Medicine and Genetics, Samsung

Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, Seoul, 06351, Republic of Korea. Tel.: +82 2 3410 2709; Fax: +82 2 3410 2719 1

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Corresponding author at: Department of Neurology, College of Medicine, Hanyang

University, 17 Haengdang-dong, Seongdong-gu, Seoul, 04763, Republic of Korea. Tel: +82 2

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2290 8371; Fax: +82 2 2296 8370 E-mail addresses: [email protected] (C.-S. Ki), [email protected] (S.H. Kim).

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These authors contributed equally to this work.

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ABSTRACT

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The TANK-binding kinase 1 (TBK1) gene has recently been identified as a novel causative

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gene of amyotrophic lateral sclerosis (ALS). This study aims to determine the frequency and

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spectrum of TBK1 variants and their functional implications in Korean patients with sporadic

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ALS (sALS). TBK1 sequences were analyzed in 129 consecutive patients with sALS using

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either multi-gene panel or exome sequencing. One frameshift (c.1414delA) and three

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missense variants of uncertain significance (VUS) in TBK1 were found in four patients each.

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In vitro functional studies revealed that the c.1414delA (p.Ile472Serfs*8) variant was

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associated with reduced mRNA expression of TBK1. Moreover, protein expression of this

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variant in patient-derived fibroblasts disrupted binding to autophagy adaptor proteins and

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inhibited the function of TBK1 in HEK293T cells. In contrast, the three other missense VUS

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showed normal mRNA expression and no abnormalities in protein function. Based on these

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findings, the frequency of pathogenic TBK1 variants in Korean sALS patients was estimated

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to be 0.8% (1/129). In conclusion, pathogenic variants in TBK1 are rare, but could be

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responsible for sALS in a small number of Korean patients.

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Keywords: amyotrophic lateral sclerosis; ALS; gene; pathogenic; TBK1; variant

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1. Introduction

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Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder

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characterized by progressive loss of cortical, bulbar, and spinal motor neurons over a period

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of approximately 3 to 5 years from symptom onset (Rowland and Shneider, 2001).

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Approximately 5-10% of patients are familial (fALS) cases, while the remaining 90% are

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apparently sporadic (sALS) cases (Rowland and Shneider, 2001).

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To date, a number of genetic loci and disease-causing genes have been reported to be associated with ALS, including SOD1, FUS, TARDBP, OPTN, SQSTM1, VCP, and

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hexanucleotide expansion of C9orf72. However, pathogenic variants in these genes have been

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identified in only two-thirds of all fALS cases and about 11% of sALS cases (Renton et al.,

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2014). An intronic hexanucleotide expansion in C9orf72 was identified in 9p21-linked ALS

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and frontotemporal lobe dementia (FTD), which is the most common cause of ALS in people

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of European ancestry. Specifically, this expansion accounted for about 40% of all fALS and

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about 7% of all sALS cases (DeJesus-Hernandez et al., 2011; Renton et al., 2011).

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Recently, TANK-binding kinase 1 (TBK1) gene was identified as a novel causative gene of

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ALS (Cirulli et al., 2015; Freischmidt et al., 2015). TBK1 encodes a protein related to

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autophagy, specifically the removal of stress granules, and interacts with OPTN, SQSTM1,

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and VCP, all of which have been previously reported as causative genes of ALS. Pathogenic 4

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variants in the C-terminal TBK1 coiled-coil domain render TBK1 unable to bind to

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optineurin. In addition, TBK1, OPTN, and SQSTM1 have functions in the NF- B pathway

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associated with innate immunity and neuroinflammation (Freischmidt et al., 2015; Weidberg

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and Elazar, 2011).

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In Korean patients with ALS, the C9orf72 repeat expansion has not been identified. Instead, SOD1 and FUS appear to be the two major genes involved in ALS (Jang et al., 2013; Kim et

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al., 2014; Kim et al., 2016; Kwon et al., 2012). In this study, we aimed to determine the

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frequency and spectrum of TBK1 variants in Korean patients with ALS. We also performed in

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vitro functional studies of different TBK1 variants.

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2. Methods

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2.1. Subjects

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Patients with ALS were enrolled from a cohort at the ALS Clinic in the Neurology

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Department of Hanyang University Hospital in Seoul, Korea, between November 2014 and

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July 2015. The diagnosis of ALS was based on the El Escorial revised criteria (Brooks et al,

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2000). Patients with clinically definite, probable, probable laboratory-supported, or possible

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ALS were recruited for this study. During this period, most cases were sALS so we decided 5

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to include only sALS cases in this study. Therefore, a total of 129 Korean patients with sALS

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were enrolled. Four of the 129 patients had concomitant FTD (ALS-FTD), two patients had

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behavioral variant FTD (bv-FTD), and another two had semantic dementia (SD). We

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reviewed the patient medical records for clinical characteristics including age, sex, family

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history of ALS, region of symptom onset, initial ALS functional rating scale revised

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(ALSFRS-R) score, and calculated progression rate (delta FS, [(48-ALSFRS-R score at

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visit)/duration from onset to visit (month)]). Hexanucleotide repeat expansion of C9orf72 was

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tested by repeat-primed PCR in all patients, as previously described (Jang et al., 2013). The

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mean onset age was 55.8±10.0 years (range 23-79 years). Eighty-one patients (62.8%) were

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male and 48 (37.2%) were female. Ninety-seven patients (75.2%) had spinal-onset ALS and

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30 (23.2%) had bulbar-onset ALS; of the remaining patients (n=2, 1.5%), one had

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respiratory-onset and the other had axial-onset ALS. The mean ALSFRS-R score at the first

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visit was 39.1±6.3 (range 4–47). The mean delta FS at diagnosis was 0.78±0.89 (range 0.06–

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8.33). This study was approved by the Institutional Review Boards of Hanyang University

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Hospital (#HYI-10-01-3) and Samsung Medical Center (#2013-04-131-002). All participants

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provided written informed consent as per the approved study protocol.

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2.2 Genetic analysis

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Genomic DNA was extracted from peripheral blood leukocytes using a Wizard Genomic

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DNA purification kit according to the manufacturer’s instructions (Promega, Madison, WI).

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Variants in TBK1 and other ALS-related genes were screened by next-generation sequencing

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(NGS). Sequencing libraries were prepared according to the manufacturer’s instructions

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using either a TruSightTM One Sequencing Panel (Illumina Inc., Sand Diego, CA) or an

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Agilent SureSelect all Exon kit 50Mb (Agilent, Santa Clara, CA). The flow cell was loaded

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on either a MiSeq or a NextSeq 500 sequencing system (Illumina) for sequencing with

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2x100bp read lengths. Reads were mapped to the GRCh37/hg19 build using the Burrows-

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Wheeler Aligner (BWA), and variants were called using GATK software. We filtered out all

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variants with allele frequencies >0.01 based on various public databases, including the

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Exome Aggregation Consortium (http://exac.broadinstitute.org/) database and the Korean

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Reference Genome Database (http://152.99.75.168/KRGDB/). Sanger sequencing validation

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was performed for all pathogenic variants in ALS-related genes and variants of uncertain

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significance (VUS) in TBK1 using primers designed by the authors.

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The Sorting Intolerant From Tolerant (SIFT) (Ng and Henikoff, 2003) and Polymorphism

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Phenotyping 2 (PolyPhen-2 v2.1) (Adzhubei et al., 2010) servers were used to predict the

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effects of nonsynonymous variants on protein structure, function, phenotype, and sequence

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conservation. In PolyPhen-2, a variant is classified as “probably damaging” if it has a

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probabilistic score above 0.85 and as “possibly damaging” if it has a score above 0.15; the 7

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remaining variants are classified as benign. Using SIFT, scores ranging from 0 to 1 are

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obtained to represent the normalized probability that a particular amino acid substitution will

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be tolerated. SIFT predicts that substitutions with scores less than 0.05 are deleterious. To

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identify evolutionarily constrained regions, multiple sequence alignments and rates of

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evolution were estimated using Genomic Evolutionary Rate Profiling (GERP) (Davydov et al., 2010).

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2.3 In vitro functional study

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We quantified TBK1 mRNA and protein expression levels in patient-derived fibroblasts

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harboring three variants: c.1414delA (p.Ile472Serfs*8), c.1150C>T (p.Arg384Trp), and

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c.1424T>C (p.Ile475Thr). Fibroblasts with the c.1426G>A (p.Glu476Lys) variant were not

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available. Cell culture and immunoblotting were performed as described previously (Lim et

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al., 2016) and quantitative real-time polymerase chain reaction (qRT-PCR) was performed

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using the primers from a previous study (Freischmidt et al., 2015). In addition, we examined

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the interactions between TBK1 variants and binding partners via a GST pull-down assay and

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analyzed IFN activity using a luciferase reporter assay to identify loss-of-function variants as

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described previously (Freischmidt et al., 2015). Comprehensive experimental procedures

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were described in the Supplementary information.

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2.4. Statistical analysis

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Statistical analysis was performed using Student’s t-test. All analysis was performed in

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GraphPad Prism Version 5.0 software for Mac OS X (GraphPad Software, San Diego, CA,

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USA).

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3. Results

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3.1. Identification of TBK1 and other gene variants

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The mean read depth of TBK1 exceeded 85x, and over 99% of the TBK1 coding sequence was covered by at least 10 independent sequence reads across all samples. Among the 129

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Korean patients with sALS, we identified four TBK1 variants, including one frameshift

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(c.1414delA) and three missense variants, and each of these was identified in different

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patients (Table 1). All missense variants were found to affect protein structure according to in

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silico analyses with Polyphen-2 or SIFT. Two missense variants (c.1150C>T and c.1426G>A)

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had matching rs numbers in the dbSNP database (rs753595352 and rs755602104,

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respectively). The allele frequencies of the TBK1 variants were lower than 0.001 or not

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reported in public databases, including ExAC and KRGDB (Table 2). Among other ALS

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genes, one pathogenic variant (c.260A>G; p.Asn87Ser) was identified in SOD1.

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3.2 Clinical features of sporadic ALS with TBK1 variants

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The demographic and clinical characteristics of the four sALS patients with TBK1 variants

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are summarized in Table 3. Patient MND-1536, who carried the TBK1 c.1414delA

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(p.Ile472Serfs*8) variant, gradually developed dysarthria and dysphagia at the age of 53

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years. Because of longstanding predominant bulbar signs including severe dysarthria, tongue

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atrophy, and swallowing difficulty, in addition to his mild limb weakness, his diagnosis was

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delayed until 34 months after symptom onset. At the time of diagnosis, upper motor neuron

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signs in the upper and lower limbs and mild distal hand muscle wasting were also present.

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His EMG findings showed widespread active denervation potentials with fasciculations in the

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upper limbs and bulbar muscles, and their motor unit potentials during volitional contraction

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showed long-duration large amplitude polyphasic patterns, suggesting signs of chronic

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denervation. His diagnosis was compatible with clinically probable ALS- laboratory-

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supported based on the revised El Escorial criteria. Despite more progressive bulbar

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symptoms, his cognition was well maintained, and he exhibited no difficulty in expressing

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himself in writing. Due to severe difficulties in swallowing, gastrostomy was performed 46th

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months after symptom onset. He had neither glaucoma nor a familial history of motor neuron

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diseases or dementia.

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3.3. TBK1 expression and function in patient-derived fibroblasts

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We next quantitated TBK1 mRNA and protein expression levels in patient-derived

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fibroblasts harboring one of the three variants of c.1414delA (p.Ile472Serfs*8), c.1150C>T

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(p.Arg384Trp), and c.1424T>C (p.Ile475Thr). Fibroblasts with the c.1426G>A (p.Glu476Lys)

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variant were unavailable. Compared with control fibroblasts, the mRNA level of TBK1 was

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reduced approximately 50% in fibroblasts with the c.1414delA (p.Ile472Serfs*8) variant.

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Consistent with this finding, the level of TBK1 protein expression was also reduced. The

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other two missense variants, c.1150C>T (p.Arg384Trp) and c.1424T>C (p.Ile475Thr),

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showed normal mRNA and protein expression levels (Fig. 1a, b).

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Activated TBK1 phosphorylates IRF3, resulting in IRF3 dimerization and subsequent

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nuclear translocation. This constitutes an essential step in the activation of the IFN pathway

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(Chau et al., 2008). The level of phospho-IRF3 was decreased only in fibroblasts with the

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c.1414delA (p.Ile472Serfs*8) variant (Fig. 1b).

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3.4. Functional analysis of TBK1 variants in HEK293T cells 11

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TBK1 is known to interact with both OPTN and IRF3 (Freischmidt et al., 2015). To

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further investigate the effects of TBK1 variants, we examined the interactions between these

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variants and their binding partners via a GST pull-down assay. HEK293T cells were

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transiently transfected with GFP-tagged wild-type or variant forms of TBK1, and the

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expression levels of the TBK1 variants were analyzed by immunoblotting. When the wild-

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type version and the three missense variants (p.Arg384Trp, p.Ile475Thr, p.Glu476Lys) of

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TBK1 were expressed, all bands migrated at the same size as full-length TBK1. However, the

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frameshift variant (p.Ile472Serfs*8) migrated at apparently different molecular weight (Fig.

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2a). This frameshift variant lacked a functional full-length TBK1 (predicted molecular weight

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110 kDa). The band for the predicted truncated protein was observed to migrate with an

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apparent molecular weight of approximately 80 kDa.

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Next, whole cell lysates of HEK293T cells transiently expressing different TBK1 variants

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were subjected to pull-down assays with GST-OPTN or GST-IRF3, and bound proteins were

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analyzed. The p.Ile472Serfs*8 variant did not bind to either OPTN or IRF3, while the three

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missense variants (p.Arg384Trp, p.Ile475Thr, and p.Glu476Lys) showed normal binding (Fig.

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2a).

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Lastly, we sought to identify loss-of-function (LoF) variants that disrupt IFN activity using a luciferase reporter assay. Compared with wild-type TBK1, the p.Ile472Serfs*8 12

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variant lost the ability to suppress IFN promoter activation (Fig. 2b). Taken together, our

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results indicate that the p.Ile472Serfs*8 variant abrogates binding to autophagy adaptor

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proteins and inhibits the function of TBK1, an important step in IFN signaling.

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4. Discussion

We evaluated the frequency and spectrum of TBK1 variants in 129 Korean patients with

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sALS. Of the one frameshift and three missense variants, only the c.1414delA

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(p.Ile472Serfs*8) variant exhibited disrupted TBK1 function. Thus, the overall frequency of

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the TBK1 pathogenic variant was estimated to be 0.8% (1/129). In addition, one pathogenic

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variant in SOD1 was found in one patient with sALS, yielding a frequency of 0.8%. This

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frequency is comparable to the findings of a previous study (1.2%) of Korean patients with

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sALS (Kwon et al., 2012).

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TBK1 was newly identified as a causative gene in patients with ALS-FTD spectrum

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disorders. TBK1 is involved in the autophagy and neuroinflammation pathways and has been

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linked to core proteins in these pathways, such as UBQLN2, VCP, OPTN, and SQSTM1

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(Cirulli et al., 2015). These pathways appear to be involved in the formation of cellular

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inclusions, which plays a key role in neurodegenerative disorders (Freischmidt et al., 2015;

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Ramaswami et al., 2013). 13

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LoF variants in TBK1 have been reported to be responsible for 0.4-4% of all ALS cases (Cirulli et al., 2015). In an Australian ALS cohort, only one truncated TBK1 variant

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(c.1197delC) was found in a Chinese patient with fALS. However, this variant was not found

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in any patients of European ancestry, suggesting that TBK1 is a rare cause of fALS in

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Australia (Freischmidt et al., 2015; Williams et al., 2015). In addition, one nonsense variant

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was identified at a low frequency (0.5%, 1/207) in a Taiwan population (Tsai et al., 2016). In

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a French population, five LoF variants from four patients with ALS-FTD and one patient with

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ALS were found to have higher frequencies in patients with familial ALS-FTD (10.4%) than

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isolated ALS (0.9%) (Le Ber et al., 2015). On the other hand, a study with a Belgian ALS-

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FTD cohort reported 11 patients carrying a LoF variant of TBK1, with an overall frequency of

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1.7% (11/629) and a frequency of 3.4% in patients with ALS (5/147). Thus, this variant was

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the second most common cause of ALS in Belgian patients, after C9orf72 repeat expansion

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(Gijselinck et al., 2015). In this study, the LoF variant explained 0.8% (1/129) of all sALS

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cases, a somewhat lower frequency.

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Approximately half of all Caucasian patients with ALS carrying a TBK1 variant exhibit

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symptoms of cognitive impairment such as mild cognitive impairment, dementia, or FTD

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(Freischmidt et al., 2015). In our study, the Korean patient with sALS carrying LoF TBK1

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variant did not exhibit cognitive impairment, although we should note that the sample size of

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our study was small. TBK1 variants were not found in four Korean patients with ALS-FTD in

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our study. TBK1 contains four domains, including an N-terminal kinase domain (KD), an ubiquitin-

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like domain (ULD), an a-helical scaffold dimerization domain (SDD), and a C-terminal

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domain (CTD). The SSD domain interacts with the KD, the ULD, and the C-terminal ends of

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the SSD, thus enabling TBK1 dimerization. Of these interactions, the interaction between the

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C-terminal ends is the main interface for TBK1 function (Larabi et al., 2013). Interestingly,

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the c.1414delA (p.Ile472Serfs*8) variant is located within the C-terminal region of the SSD.

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Through in vitro functional studies, we demonstrated reduced TBK1 protein expression,

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impaired TBK1 function in patient-derived fibroblasts, and disrupted binding ability to

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autophagy adaptor proteins in HEK293T cells expressing this variant. Therefore, we

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hypothesize that the p.I472Sfs*8 variant hinders TBK1 dimerization, thereby abrogating its

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function.

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In conclusion, pathogenic variants in TBK1 gene exist in Korean patients with sALS, albeit

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at low frequencies. Our data indicate that this gene could be responsible for sALS in some

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Korean patients.

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Disclosure statement The content of this article represents original work that has not been previously published

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and is not under consideration for publication elsewhere. There are no conflicts of interest to

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declare.

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Acknowledgement

This study was supported by grants from the Korean Health Technology R&D Project,

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Ministry for Health, Welfare & Family Affairs, Republic of Korea (HI12C0135) and the

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Basic Science Research Program through the National Research Foundation of Korea (NRF)

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funded by the Ministry of Science, ICT & Future Planning (NRF-2014R1A1A3052516).

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Table 1. TBK1 variants identified in the 129 patients with sporadic ALS

(hg19/GRch37)

MND-1447

MND-1448

MND-1536 *

Chr12:64,878,240

Chr12:64,882,350

Chr12:64,882,352

Chr12:64,882,340

9

12

12

12

change*

Amino acid change

c.1150C>T

c.1424T>C

p.Arg384Trp

c.1426G>A

c.1414delA

GERP

PolyPhen-2

SIFT

(Probabilistic score)

(Tolerance index)

Probably damaging

4.17

(1.00)

(0.00)

Benign

Deleterious 4.08

(0.002)

(0.00)

Benign

Deleterious

(0.025)

(0.00)

N/A

N/A

p.Glu476Lys

p.Ile472Serfs*8

4.36

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Nucleotides are numbered according to the reference cDNA sequence, GenBank accession number NM_013254.3.

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score

Deleterious

p.Ile475Thr

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MND-1192

Nucleotide

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Exon

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Genomic coordinates

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Patient ID

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In-silico analysis

N/A

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Table 2. Allele frequencies of TBK1 variants in public databases

Allele frequency

Patient ID

Nucleotide change

*

Amino acid

rs number

change

†

KRGDB‡

c.1150C>T

p.Arg384Trp

rs753595352

MND-1447

c.1424T>C

p.Ile475Thr

N/A

MND-1448

c.1426G>A

p.Glu476Lys

MND-1536

c.1414delA

p.Ile472Serfs

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MND-1192

rs755602104

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N/A

0.00004

None

None

None

None

None

0.00001

0.0008

None

None

None

None

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*Nucleotides are numbered according to the reference cDNA sequence, GenBank accession number NM_013254.3. †

ExAC, Exome Aggregation Consortium (http://exac.broadinstitute.org/, access on 27/09/2016).

KRGDB, Korean Reference Genome Database (http://152.99.75.168/KRGDB/, access on 27/09/2016).

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control (n=202)

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ExAC

In-house

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Amino acid

Duration from

Sex

onset

onset

change

(year)

to latest visit

ALSFRS-R

delta-FS

(years)

p.Arg384Trp

M

40

41

22

42

MND-1447

p.Ile475Thr

F

55

57

25

40

MND-1448

p.Glu476Lys

F

56

58

41

MND-1536

p.Ile472Serfs

M

53

56

47

0.75

Cognitive

Diagnostic certainty impairment

Lower limb

Definite

No

0.42

Upper limb

Probable

No

36

0.43

Upper limb

Probable

No

37

0.32

Bulbar

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Region of onset

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Patient ID

Age of Age

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Table 3. Demographic and clinical characteristics of patients with TBK1 variants

Probable -laboratory No

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Fig. 1. Quantification of TBK1 mRNA and protein expression levels in patient-derived fibroblasts.

(a) Quantitative RT-PCR analysis of TBK1 mRNA expression in fibroblasts from healthy

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control subjects and from subjects harboring TBK1 variants. Expression levels were normalized to that of TBP. Values are expressed as mean ± SEM. *P < 0.001; Unpaired t-test.

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(b) Western blot analysis of TBK1 expression in cultured fibroblasts from healthy control subjects and from subjects harboring TBK1 variants. In patient-specific fibroblasts, the kinase

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activity of the different TBK1 variants was assessed by examining phosphorylation (Ser396) of total IRF3. β-actin is shown as a loading control. Western blots were repeated three times.

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Fig. 2. Functional analysis of TBK1 variants.

In vitro functional analysis of the four TBK1 variants. (a) Interactions between TBK1+OPTN and TBK1+IRF3 as assessed by a GST pull-down assay. Lysates from HEK 293T cells

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expressing GFP-TBK1-WT or the indicated variants were incubated with GST-OPTN or GST-IRF3. Equal GST-OPTN and GST-IRF3 protein levels were confirmed by Ponceau S

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staining. Cell lysates and bound proteins were analyzed by Western blotting using anti-GFP

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antibody. The arrowhead in (a) marks a band corresponding to the migration pattern of p.Ile472Serfs*8, which is a frameshifted version of TBK1. Western blots were repeated three times. (b) Luciferase activity in HEK293T cells transiently transfected with an IFN-β reporter plasmid and a GFP-tagged wild-type plasmid or a variant TBK1 plasmid for 24 h. Firefly luciferase activity served as an internal control. Data are presented as the mean ± SEM of three independent experiments. *P < 0.001; unpaired t-test. 21

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sporadic amyotrophic lateral sclerosis (sALS).

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We performed genetic and functional analysis of TBK1 gene in 129 Korean patients with


We identified four novel TBK1 variants comprised of one frameshift and three missense

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variants and each of these was observed in four different patients.


Functional analysis revealed that only frameshift variant showed functional impairment

function.

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while the other three missense variants showed normal mRNA expression and protein


Therefore, the frequency of pathogenic TBK1 variants in Korean sALS patients was

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estimated to be 0.8% (1/129).


Pathogenic variants in TBK1 are rare, but could be responsible for sALS in a small

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number of Korean patients.

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Disclosure statement

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The content of this article represents original work that has not been previously published or in consideration of publication elsewhere. There are no conflicts of interest

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Neurobiology of Aging by all contributing authors.

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to declare. The manuscript has been read and approved for submission to the

Aknowledgements

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This study was supported by grants from the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (HI12C0135) and the

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Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-

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2014R1A1A3052516).