Gijselinck et al._Supplementary DataNEUROLOGY/2015/667717
Loss of TBK1 is a frequent cause of frontotemporal dementia in a Belgian cohort
Gijselinck, Ilse a b, Van Mossevelde, Sara a b, van der Zee, Julie a b, Sieben, Anne a b c, Philtjens, Stéphanie a b, Heeman, Bavo a b, Engelborghs, Sebastiaan b d, Vandenbulcke, Mathieu e f; De Baets, Greet g, Bäumer, Veerle a b, Cuijt, Ivy a b, Van den Broeck, Marleen a b, Mattheijssens, Maria a b, Peeters, Karin a b, Rousseau, Frederic g, Vandenberghe, Rikf h, De Jonghe, Peter a b i, Cras, Patrick b i, De Deyn, Peter P. b d #, Martin, Jean-Jacques b, Cruts, Marc a b *,Van Broeckhoven, Christine a b * on behalf of the BELNEU consortium $.
a Department of Molecular Genetics, VIB, Antwerp, Belgium
b Institute Born-Bunge, University of Antwerp, Antwerp, Belgium
c Department of Neurology, University Hospital Ghent and University of Ghent, Ghent, Belgium
d Department of Neurology and Memory Clinic, Hospital Network Antwerp Middelheim and HogeBeuken, Antwerp, Belgium
e Brain and Emotion Laboratory, Department of Psychiatry, University of Leuven, Leuven, Belgium
f Department of Neurology, University Hospitals Leuven, Gasthuisberg, Leuven, Belgium
g SWITCH laboratory, VIB, University of Leuven, Leuven, Belgium
h Laboratory for Cognitive Neurology, Department of Neurology, University of Leuven, Leuven, Belgium
i Department of Neurology, Antwerp University Hospital, Edegem, Belgium
# Peter P. de Deyn is also affiliated to the Department of Neurology and Alzheimer research Center, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands
$ Belgium Neurology (BELNEU) consortium members are listed in the co-investigator appendix
*Corresponding authors:
Prof. Dr. Christine Van Broeckhoven PhD DSc
Neurodegenerative Brain Diseases Group
VIB Department of Molecular Genetics
University of Antwerp - CDE
Universiteitsplein 1, 2610 Antwerp, Belgium
Tel: +32 3 265 1101
E-mail:
Prof. Dr. Marc Cruts PhD
Neurodegenerative Brain Diseases Group
VIB Department of Molecular Genetics
University of Antwerp - CDE
Universiteitsplein 1, 2610 Antwerp, Belgium
Tel: +32 3 265 1033
E-mail:
Key words: Frontotemporal lobar degeneration, FTLD-ALS, TBK1, loss-of-function, mutations
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SUPPLEMENTARY MATERIALS AND METHODS
Participants
The Belgian hospital-based cohort consisted of 460 patients with FTD, 147 with ALS and 22 with concomitant FTD and ALS (FTD-ALS) (1, 2). The FTD and ALS patients were collected from 1995 onwards through an ongoing multicentre collaboration of neurology departments and memory clinics in Belgium (BELNEU consortium). Additional patients were included who had initially been referred to our Diagnostic Service Facility for medical genetic testing. Patients were diagnosed using a standard protocol and established clinical criteria (1-5). For ALS patients, neuropsychological testing was not done. Post mortem neuropathological analysis confirmed diagnosis in 25 FTD, three FTD-ALS and six ALS patients. A positive familial history, i.e. at least one first-degree relative with a FTD-ALS spectrum disease, was recorded in 132 FTD (28.7%), four FTD-ALS (36.4%) and 18 AS (12.2%) patients. Of these familial index patients, 93 FTD (70.5%), one FTD-ALS (25.0%) and seven ALS (38.9%) patients were not explained by mutations in the known FTLD, ALS and AD genes (MAPT, GRN, C9orf72, VCP, CHMP2B, FUS, TARDBP, PSEN1, PSEN2, APP, PRNP, SOD1). Of the one unexplained FTD-ALS multiplex family (family DR158) DNA, lymphoblast cell lines, serum and plasma was collected of 38 individuals including four patients (Figure 1, Table 1).
The control cohort consisted of 1044 age-matched individuals from the same geographical Flanders-Belgium region, free of personal and familial history of neurodegenerative or psychiatric diseases and with a Mini Mental State Examination score >26.
All participants provided written informed consent for participation in clinical and genetic studies. Clinical study protocols and informed consent forms were approved by the local medical ethics committees of the collaborating clinical centers. Genetics study protocol and informed consent forms were approved by the medical ethics committees of the University of Antwerp.
TBK1 Mutation screening
19 coding exons of TBK1 were amplified in multiplex PCR reactions using the Multiplex Amplification of Specific Targets for Resequencing (MASTR) (Multiplicom N.V., Niel, Belgium) technology. Primers for multiplexing were designed using the mpcr primer design tool (Multiplicom N.V.). Targets were amplified in highly multiplexed PCR reactions and unique dual indices were added to these amplicons, resulting in up to 1536 uniquely barcoded samples (6). After equimolar pooling, the barcoded samples were sequenced on the MiSeq platform using the MiSeq V2 chemistry (250 bp paired-end reads, Illumina, San Diego, CA, USA). After sample demultiplexing, sequence reads were mapped using the Burrows-Wheeler Aligner (BWA) (7) to a minigenome consisting of the combined amplicon sequences extracted from the human genome reference sequence hg19. Sequence variants were called using the Genome Analysis Toolkit (GATK) (8, 9) and SAM (Sequence Alignment/Map) tools (10), annotated using the GenomeComb variant annotation pipeline (11) and visualized in IGV (12, 13). Exon 4, which failed in this setup, was PCR amplified in simplex followed by Sanger sequencing using the BigDye® Terminator Cycle Sequencing kit v3.1 (Applied Biosystems) on an ABI3730 automated sequencer (Applied Biosystems). Variants identified in the mutation analysis were validated by PCR–based amplification of genomic DNA followed by Sanger sequencing.
We predicted the pathogenic effect of rare coding variants using two conservation scoring programs SIFT (Sorting Intolerable From Tolerable) (14)and ConSurf(15). The effect on stability and dimerization was evaluated in silico by FoldX(16-18).
Gene dosage analysis
A selection of 270 samples was analyzed for copy number changes using the Multiplex Amplicon Quantification (MAQ) technique (Multiplicom N.V., Niel, Belgium), consisting of a multiplex PCR amplification of fluorescently labeled test and reference amplicons, followed by fragment analysis on an ABI 3730 DNA analyzer (Applied Biosystems). Eighteen test amplicons in 18 exons of TBK1 and 14 reference amplicons located at randomly selected genomic positions outside known CNVs were simultaneously PCR-amplified on 20 ng genomic DNA. Peak areas of the test amplicons were normalized to these of the reference amplicons. Comparison of normalized peak areas between a patient and control individuals resulted in a dosage quotient (DQ) for each test amplicon, calculated by the MAQ software (MAQs) package. DQ values below 0.75 or above 1.25 were considered indicative of a heterozygous deletion or duplication respectively.
STR genotyping
STR markers flanking TBK1 were selected from the Marshfield gender-averaged genetic map ( or from the Tandem Repeat Finder (11) implemented in the UCSC genome browser ( Primer pairs for each selected marker were designed with the mpcr primer design tool (Multiplicom N.V.) (primers available upon request) and genotyped in 6 different index patients carrying the p.Glu643del mutation and their relatives for haplotype sharing. The resulting PCR products were separated and analyzed on an ABI 3730 automated sequencer (Applied Biosystems) and genotypes were assigned using in-house developed TracI genotyping software (
Transcript analysis
Transcript analysis was performed for patients with a TBK1 mutation and control individuals without mutation using lymphoblast cells and frontal cortical brain tissue (Table 2). RNA and proteins from lymphoblast cells were extracted at two different time points during cell culturing to validate that the results are not dependent on the number of passages of the cell lines. For quantification of TBK1 lymphoblast and brain transcripts, we used semi-quantitative real-time PCR (qPCR) using SYBR Green assays on the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). We designed a qPCRamplicon spanning exon 19-20 (5’- CATGACCCCAATTTATCCAAGTTC-3’ and 5’- CATCTCTTCCTTTAATTTCTTCATACCA-3') detecting the TBK1refgene isoform NM_013254 using PrimerExpress Software (Applied Biosystems) and quantified against two housekeeping genes YWHAZ and TBP for lymphoblast RNA and HPRT, GAPDH, SDHA for brain RNA. Total RNA was isolated using the Ribopure Kit for total RNA isolation (Ambion) and treated with DNase (Turbo DNase Kit, Ambion). First-strand cDNA was synthesized from total RNA with oligodT and random hexamers primers using the SuperScript III First-Strand Synthesis System for RT-PCR kit (Invitrogen, Carlsbad, CA USA). cDNA of these samples was amplified in triplicate using the universal amplification protocol (Applied Biosystems) as previously described (19). Relative expression levels were calculated by comparing normalized quantities between patients and control samples using qbase+ software (Biogazelle).
Nonsense-mediated mRNA decay (NMD) analysis of the p.Ser398Profs*11 mutation in patient DR1123 and the p.Gly272_Thr331del mutation in patient DR189 was performed on cDNA prepared from lymphoblast cells treated with 100 μg/ml cycloheximid for 4 hours prior to mRNA isolation. PCR was performed on cDNA of treated and non-treated samples with primers amplifying the complete coding region of the TBK1 transcript (5’- agccggaagtgtcctgagt-3’ and 5’-gccaccatccatggttaaag-3’) or parts of it (primers upon request). The resulting PCR products were Sanger sequenced to determine the relative abundance of transcribed alleles based on the presence of the coding mutation or a coding SNP rs7486100 in exon 8 and to evaluate the presence or absence of aberrant transcripts. Genotypes of cDNA sequences were compared with gDNA sequences. Amplified cDNA of the p.Gly272_Thr331del mutation carrier was cloned in a pCR™4-TOPO® TA Vector using the Topo-TA cloning kit for sequencing (Life Technologies) and propagated in E. coli cells to determine the sequence of the different PCR products.
Protein analysis
Protein lysates for western blot were prepared with 0.1% RIPA and sonicated on ice, cleared at 20 000 g for 15 min at 4°C and supernatants used for immunoblotting. Protein concentrations were measured by a BCA assay (Pierce) and 30 micrograms of protein were separated on 4-12% NupageBis-Tris gels (Invitrogen), electroblotted onto a PVDF membrane (Hybond P; Amersham Biosciences) and probed with a monoclonal antibody against TBK1 (Abcam, 1:1000, 84kDa). Immunodetection was performed with specific secondary antibodies conjugated to horseradish peroxidase and the ECL-plus chemiluminescent detection system (Amersham Biosciences).
TBK1 signal intensities were quantified against GAPDH (Genetex, 1:10000, 37kDa), which was used as an internal control protein using ImageQuantTL software (GE Healthcare Life Sciences).
Neuropathological and immunohistochemical analysis
Autopsied brains of two TBK1 patients DR1124 and DR189 were obtained using informed consents and protocols that were approved by the Ethical Committee of University of Antwerp and Antwerp University Hospital and stored in The Antwerp Biobank of the Institute Born-Bunge. After a fixation period of 8 to 16 weeks in 10% buffered formalin, 5µm slices were cut. Samples were obtained from frontal cortex, temporal neocortex (superior temporal gyrus), hippocampus, area striata, neostriatum, basal ganglia, substantianigra, thalamus, mesencephalon, pons, medulla oblongata, cerebellum and in addition spinal cord of DR1124. Sections were deparaffinized, rehydrated and pretreated with citric acid 0.1M. Immunohistochemical analysis was performed with anti-ubiquitin antibody (Dako, Glostrup, Denmark), AT8 against hyperphosphorylated tau (Innogenetics, Zwijnaarde, Belgium), 4G8 against β-amyloid (Signet, Dedham, Massachusetts), anti-FUS antibody (Sigma Aldrich, St Louis), anti-TDP-43 antibody (Proteintech Group Inc, Chicago, Illinois). Additionally, immunohistochemistry was performed with anti-p62 antibody (DB Transduction Laboratories) and polyclonal anti-TBK1 antibody (Sigma, 1:200). Sections were counterstained with hematoxylin and images were taken on an Axioskop 50 light microscope (Zeiss) equipped with a CCD UC30 camera (Olympus Inc.).
Two controls without mutations were also stained for TBK1: a man aged 81 who died of the complications of acute paraplegia and 57 year old male patient who died of respiratory complications.
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Table e-1. Genomic and protein annotation of TBK1 mutations
Genomic mutation1 / Predicted protein2 / ProteinDomain
g.3815C>T / p.Gln2* / KD
g.14982_14984del / p.Asp167del / KD
g.29963G>T / p.Gly272_Thr331del / KD - ULD
g.33398delT / p.Ser398Profs*11 / SDD
g.43453_43454insTT / p.Ser518Leufs*32 / SDD
g.45168_45170del / p.Glu643del / SDD
g.28063G>T / p.Arg271Leu / KD
g.29841A>G / p.Lys291Glu / KD
g.29934C>T / p.His322Tyr / ULD
g.43446T>C / p.Ile515Thr / SDD
g.43505G>A / p.Ala535Thr / SDD
Notes: 1gDNA numbering relative to NC_000012.12; 2protein numbering according to NP_037386.1 isoform; KD: kinase domain; ULD: ubiquitin-like domain; SDD: alpha-helical scaffold dimerization domain
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Table e-2. Detailed clinical characteristics of TBK1 LOF mutation carriers.
Patient ID / gender / age at onset (years) / disease duration (years) / clinical diagnosis / subtype FTLD / subtype ALS / Clinical featuresfrontal features (apathy, disinhibition) / language difficulties / memory deficits / orientation deficits / extra pyramidal symptoms / MND features / psychiatric symptoms
DR158.II.1 / F / 71 / 10 / D / no detailed information / +
DR158.II.2 / F / 86 / 4 / D / no detailed information
DR158.III.1 / M / 69 / 3 / ALS / - / spinal / + / + / +
DR158.III.2 / M / 69 / 6 / FTD / bvFTD / - / + / + / + / + / + / +
DR158.III.3 / M / 70 / 3 / D / ? / - / + / + / + / + / +
DR158.III.4 / M / 63 / >3 / FTD / bvFTD / - / + / +
DR158.III.5 / F / 66 / 6 / FTD / bvFTD / - / + / + / + / + / +
DR158.III.6 / F / 61 / 13 / FTD / bvFTD / - / + / + / + / + / +
DR158.III.7 / F / 63 / 6 / D / no detailed information
DR158.III.8 / F / 62 / 11 / FTD-ALS / bvFTD / spinal / + / + / + / +
DR158.III.9 / F / 73 / 11 / D / ? / - / + / + / + / +
DR158.III.10 / F / 82 / 4 / D / no detailed information / +
DR158.III.11 / M / 63 / 1 / ALS / no detailed information
DR1120 / F / 56 / 4 / FTD / bvFTD / - / + / + / + / + / +
DR1127 / M / 60 / 1 / ALS / - / ? / no detailed information
DR189 / M / 48 / 2 / FTD / bvFTD / - / +
DR1123 / M / 59 / > 5 / ALS / - / bulbar / +
DR1124 / F / 64 / <1 / ALS / - / bulbar / + / + / +
DR467 / F / 64 / > 9 / FTD / bvFTD / - / + / + / + / + / + / +
DR1121 / M / 70 / > 6 / FTD / PPA / - / + / + / + / + / +
DR1122 / F / 69 / > 7 / FTD / bvFTD / - / + / + / + / + / +
DR1044 / M / 63 / 3 / ALS / - / ? / no detailed information
DR663 / M / 41 / < 1 / ALS / - / bulbar / no detailed information
D: Dementia; F: female; M: male; NA: not analyzed; +: present; -: not applicable; empty field: not reported; ?: unknown
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Table e-3. Haplotype sharing between p.Glu643del mutation carriers
MARKER / Position on chr12 (Mb)* / DR467 / DR1121 / DR1044 / DR1122 / DR663.II.2 / DR663.II.1D12S1700 / 60.0 / 200-194 / 188-208 / 198-202 / 204-194 / 204-198 / 204-198
TBK1-(TA)n / 61.4 / 248-236 / 234-262 / 226-262 / 232-248 / 232-226 / 232-226
D12S1726 / 62.4 / 314-312 / 312-312 / 312-310 / 312-308 / 312-308 / 312-308
D12S329 / 63.1 / 160-154 / 148-156 / 154-154 / 150-162 / 150-154 / 150-154
TBK1-(GT)n / 64.5 / 130-128 / 130-142 / 130-132 / 128-128 / 128-128 / 128-128
TBK1 p.Glu643del / 64.9 / del-wt / del-wt / del-wt / del-wt / del-wt / del-wt
D12S1610 / 65.0 / 352-352 / 352-350 / 352-350 / 350-352 / 350-354 / 350-354
D12S1649 / 65.6 / 280-274 / 280-284 / 280-278 / 282-280 / 282-280 / 282-280
D12S75 / 66.5 / 344-344 / 344-346 / 344-346 / 348-346 / 348-348 / 348-348
D12S1601 / 67.5 / 406-406 / 406-400 / 406-406 / 402-408 / 402-404 / 402-404
Genomic position of TBK1-(TA)n: chr12:61,436,543-61,436,594
Genomic position of TBK1-(GT)n: chr12:64,495,089-64,495,131
*According to the human reference sequence (GRCh37)
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Figure e-1. Location of mutations on the TBK1 crystal structure
Single amino acid deletions and missense mutations observed in patients (Table 2) are shown on the crystal structure of TBK1. In blue, the two missense mutations that were also found in 1 to 2 control individuals. Three functional domains can be distinguished: the kinase domain (KD), the ubiquitin-like domain (ULD) and the scaffold dimerization domain (SDD).
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Figure e-2. Transcript and protein analysis of TBK1 missense mutations
(A) qPCR quantification of TBK1 transcripts from lymphoblasts missense mutation carriers (grey) compared to controls without mutation (black) normalized to different housekeeping genes.Error bars represent the standard deviation between the different measurements.
(B) Western Blot analysis of protein extracts from lymphoblasts of missense mutation carriers compared to controls without mutation. The upper band represents TBK1 (84kDa) and the lower band the housekeeping protein GAPDH (37kDa). The graphs below show the quantification in controls (black) and patients (grey) of the TBK1 signal normalized to the signal of GAPDH. Error bars represent the standard deviation between the different measurements. (P: patients only; P+C: found in patients and controls with MAF<1%)
A
B
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Figure e-3. Distribution of different mutations in the total and familial FTD, ALS and FTD-ALS subpopulations
FTD
TOTALFAMILIAL
ALS
TOTALFAMILIAL
FTD-ALS
TOTALFAMILIAL
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