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Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia

Abstract

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative syndrome hallmarked by adult-onset loss of motor neurons. We performed exome sequencing of 252 familial ALS (fALS) and 827 control individuals. Gene-based rare variant analysis identified an exome-wide significant enrichment of eight loss-of-function (LoF) mutations in TBK1 (encoding TANK-binding kinase 1) in 13 fALS pedigrees. No enrichment of LoF mutations was observed in a targeted mutation screen of 1,010 sporadic ALS and 650 additional control individuals. Linkage analysis in four families gave an aggregate LOD score of 4.6. In vitro experiments confirmed the loss of expression of TBK1 LoF mutant alleles, or loss of interaction of the C-terminal TBK1 coiled-coil domain (CCD2) mutants with the TBK1 adaptor protein optineurin, which has been shown to be involved in ALS pathogenesis. We conclude that haploinsufficiency of TBK1 causes ALS and fronto-temporal dementia.

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Figure 1: Summary of TBK1 mutations found in ALS patients.
Figure 2: Pedigrees of the Danish/Swedish ALS families 11 and 12 heterozygous for the p. 690-713del TBK1 mutation.
Figure 3: Adaptor protein binding and kinase activity of ALS-associated mutated TBK1.

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References

  1. Andersen, P.M. & Al-Chalabi, A. Clinical genetics of amyotrophic lateral sclerosis: what do we really know? Nat. Rev. Neurol. 7, 603–615 (2011).

    Article  CAS  Google Scholar 

  2. Weidberg, H. & Elazar, Z. TBK1 mediates crosstalk between the innate immune response and autophagy. Sci. Signal. 4, pe39 (2011).

    Article  Google Scholar 

  3. Wild, P. et al. Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth. Science 333, 228–233 (2011).

    Article  CAS  Google Scholar 

  4. Pilli, M. et al. TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation. Immunity 37, 223–234 (2012).

    Article  CAS  Google Scholar 

  5. Komatsu, M., Kageyama, S. & Ichimura, Y. p62/SQSTM1/A170: physiology and pathology. Pharmacol. Res. 66, 457–462 (2012).

    Article  CAS  Google Scholar 

  6. Maruyama, H. et al. Mutations of optineurin in amyotrophic lateral sclerosis. Nature 465, 223–226 (2010).

    Article  CAS  Google Scholar 

  7. Fecto, F. et al. SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis. Arch. Neurol. 68, 1440–1446 (2011).

    Article  Google Scholar 

  8. Li, B. & Leal, S.M. Methods for detecting associations with rare variants for common diseases: application to analysis of sequence data. Am. J. Hum. Genet. 83, 311–321 (2008).

    Article  CAS  Google Scholar 

  9. Exome Aggregation Consortium. ExAC Brower Beta. http://exac.broadinstitute.org (5 October 2014).

  10. Abhinav, K. et al. Amyotrophic lateral sclerosis in South-East England: a population-based study. The South-East England register for amyotrophic lateral sclerosis (SEALS Registry). Neuroepidemiology 29, 44–48 (2007).

    Article  CAS  Google Scholar 

  11. Uenal, H. et al. Incidence and geographical variation of amyotrophic lateral sclerosis (ALS) in Southern Germany—completeness of the ALS registry Swabia. PLoS ONE 9, e93932 (2014).

    Article  Google Scholar 

  12. Gunnarsson, L.G., Dahlbom, K. & Strandman, E. Motor neuron disease and dementia reported among 13 members of a single family. Acta Neurol. Scand. 84, 429–433 (1991).

    Article  CAS  Google Scholar 

  13. Pomerantz, J.L. & Baltimore, D. NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase. EMBO J. 18, 6694–6704 (1999).

    Article  CAS  Google Scholar 

  14. Morton, S., Hesson, L., Peggie, M. & Cohen, P. Enhanced binding of TBK1 by an optineurin mutant that causes a familial form of primary open angle glaucoma. FEBS Lett. 582, 997–1002 (2008).

    Article  CAS  Google Scholar 

  15. Goncalves, A. et al. Functional dissection of the TBK1 molecular network. PLoS ONE 6, e23971 (2011).

    Article  CAS  Google Scholar 

  16. Wong, Y.C. & Holzbaur, E.L. Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation. Proc. Natl. Acad. Sci. USA 111, E4439–E4448 (2014).

    Article  CAS  Google Scholar 

  17. Korac, J. et al. Ubiquitin-independent function of optineurin in autophagic clearance of protein aggregates. J. Cell Sci. 126, 580–592 (2013).

    Article  CAS  Google Scholar 

  18. Fingert, J.H. et al. Copy number variations on chromosome 12q14 in patients with normal tension glaucoma. Hum. Mol. Genet. 20, 2482–2494 (2011).

    Article  CAS  Google Scholar 

  19. Cirulli, E.T. et al. Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways. Science published online, doi:10.1126/science.aaa3650 (15 February 2015).

  20. Ikeda, F. et al. Involvement of the ubiquitin-like domain of TBK1/IKK-i kinases in regulation of IFN-inducible genes. EMBO J. 26, 3451–3462 (2007).

    Article  CAS  Google Scholar 

  21. Hubers, A. et al. Polymerase chain reaction and Southern blot-based analysis of the C9orf72 hexanucleotide repeat in different motor neuron diseases. Neurobiol Aging 35, 1–6 (2014).

    Article  Google Scholar 

  22. van Blitterswijk, M. et al. Evidence for an oligogenic basis of amyotrophic lateral sclerosis. Hum. Mol. Genet. 21, 3776–3784 (2012).

    Article  CAS  Google Scholar 

  23. Andersen, P.M. et al. EFNS guidelines on the clinical management of amyotrophic lateral sclerosis (MALS)—revised report of an EFNS task force. Eur. J. Neurol. 19, 360–375 (2012).

    Article  Google Scholar 

  24. Erdmann, J. et al. Dysfunctional nitric oxide signaling increases risk of myocardial infarction. Nature 504, 432–436 (2013).

    Article  CAS  Google Scholar 

  25. Andersen, P.M. et al. Phenotypic heterogeneity in motor neuron disease patients with CuZn-superoxide dismutase mutations in Scandinavia. Brain 120, 1723–1737 (1997).

    Article  Google Scholar 

  26. Akimoto, C. et al. A blinded international study on the reliability of genetic testing for GGGGCC-repeat expansions in C9orf72 reveals marked differences in results among 14 laboratories. J. Med. Genet. 51, 419–424 (2014).

    Article  CAS  Google Scholar 

  27. Li, H. Toward better understanding of artifacts in variant calling from high-coverage samples. Bioinformatics 30, 2843–2851 (2014).

    Article  CAS  Google Scholar 

  28. Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, 559–575 (2007).

    Article  CAS  Google Scholar 

  29. Abecasis, G.R., Cherny, S.S., Cookson, W.O. & Cardon, L.R. Merlin–rapid analysis of dense genetic maps using sparse gene flow trees. Nat. Genet. 30, 97–101 (2002).

    Article  CAS  Google Scholar 

  30. Barrett, J.C., Fry, B., Maller, J. & Daly, M.J. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21, 263–265 (2005).

    Article  CAS  Google Scholar 

  31. Larabi, A. et al. Crystal structure and mechanism of activation of TANK-binding kinase 1. Cell Reports 3, 734–746 (2013).

    Article  CAS  Google Scholar 

  32. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  33. Livak, K.J. & Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are indebted to the patients and their families for their participation in this project. We thank A. Knehr, N. Todt, E. Jasovskaja, B. Schmoll, A.-K. Rikardsson, A. Birve, A.-C. Nilsson and the Ulm Neurology biobank team for technical assistance. We also thank the many physicians who provided samples for this study, particularly L. Brättström (Karlskrona Hospital, Sweden), S. Saker (Généthon cell and DNA bank), S. Forlani (ICM DNA and cell bank), and E. LeGuern and C. Cazeneuve (APHP, Hôpital Pitié-Salpêtrière). We also thank P. Sarvari and D.Y.R. Stainier for early studies on the role of TBK1 in Zebrafish neurodegeneration. The authors would like to thank the Exome Aggregation Consortium and the groups that provided exome variant data for comparison. A full list of contributing groups can be found at http://exac.broadinstitute.org/about. This work was supported by grants from the German Federal Ministry of Education and Research (STRENGTH consortium, Sophia and BiomarkAPD and BMBF; 01GI0704, German network for ALS research (MND-NET), O1GI1007A Competence net neurodegenerative dementias/Frontotemporal lobar degeneration consortium (FTLDc)), the German Research Foundation (SYNERGY excellence cluster), the Charcot Foundation for ALS Research (A.C.L., J.H.W.), the virtual Helmholtz Institute “RNA-Dysmetabolismus in ALS and FTD” and the DFG-funded Swabian ALS Registry, the LOEWE Centrum for Gene and Cell therapy and LOEWE Ub-Net (I.D. and B.R.), the Swedish Research Council, the Swedish Brain Power Foundation, the Swedish Brain Research Foundation and the Ulla-Carin Lindquist Foundation, the Hållsten Research Foundation, the Swedish Association for the Neurologically Disabled, the Knut and Alice Wallenberg Foundation, by the Association pour la Recherche sur la Sclérose latérale amyotrophique et autres maladies du motoneurone (ARSla, France, contract R13132DD) and the Association française contre les myopathies (AFM, France, contract R11038DD to S.M.).

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T.M.S., P.M.A., A.C.L., J.H.W., T. Meitinger, I.D., T.W., A.F., B.R., W.R., V.S., T. Brännström, P.K., P.L., A.E.V. and D.R.T. designed experiments, and analyzed and interpreted data. K.M., N.M., E.G., U.N., M.S.F., K.M.D., D.R.T., T. Brännström, A.E.V. and P.L. performed and analyzed experiments. F.N., A.H., P.W., S. Pinto, R.P., S.M., N.M., E.B., C.D., M.-H.S., J.D., T. Meyer, A.S.W., J.W., M.d.C., M.O., A.C.L., P.M.A. and J.H.W. clinically characterized patients. J.H.W., P.M.A., T.M.S., T. Meitinger, I.D., B.R. and A.C.L. wrote the manuscript. S. Putz and T.M.B. provided patient-derived cell lines.

Corresponding authors

Correspondence to Peter M Andersen or Jochen H Weishaupt.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Quantile-quantile plots showing the expected −log10 p-values for different burden tests.

CMC tests were used to compare 251 case and 827 control exomes. The 95% confidence interval is shown in grey. This gene is located in a non-unique region.

Supplementary Figure 2 Pedigrees of ALS families 2, 3, 5, 6 and 13 with TBK1 mutations for which co-segregation with disease could be assessed (in addition to families 11 and 12, see main Figure 2).

Numbering of families refers to the numbering in Table 2. The mutation of each family is given at top of respective pedigrees.

Supplementary Figure 3 Neuropathological findings in the ALS/FTD case with TBK1 p.690-713del mutation (patient V-5 of family 11 in Fig. 2).

(a) Micrograph of a section from right temporal lobe showing microvacuolation and gliosis (Hematoxylin/Eosin). (b) Micrograph showing Type B TDP-43+ perinuclear inclusion in neurons in lamina III of the temporal lobe (polyclonal anti-TDP43, Proteintech). (c) Micrograph showing p62+ perinuclear inclusions in lamina III in the right parahippocampal gyrus (monoclonal anti-p62, BD Biosciences).

Supplementary Figure 4 TBK1 expression in patient-derived LCLs, fibroblasts and keratinocytes at mRNA and protein level.

(a) qRT-PCR measurement of TBK1 mRNA abundance in LCLs, fibroblasts and keratinocytes of 3-5 healthy controls and respective TBK1 mutant cell lines normalized to TBP. The bars indicate mean ± s.d. (b) PCR products amplified from LCL-derived cDNA using oligonucleotides spanning Exon 3 and 5 of TBK1 mRNA separated on a 1% agarose gel. The arrowhead marks a band exclusively observed in LCLs carrying the direct TBK1 splice site mutation c.358+2T>C. Sequencing of this band reveals the retention of the first 226 bp of the Intron separating Exon 4 and 5 in the mature mRNA and usage of an alternative splice site. The molecular size marker is indicated in bp. (c) PCR products amplified from fibroblast-derived cDNA using oligonucleotides spanning Exon 17/18 and 21 of TBK1 mRNA separated on a 1% agarose gel. The arrowhead marks a band exclusively observed in fibroblasts carrying the TBK1 c.2138+2T>C mutation. Sequencing of this band reveals the predicted skipping of Exon 20 in the mature mRNA. The molecular size marker is indicated in bp. (b, c) are representative images out of 2 replicates. (d, e) Western blots comparing TBK1 protein expression of LCLs (d) and fibroblasts (e) from healthy controls and respective TBK1 mutant cell lines. Western blots were repeated three times The arrowhead in (e) marks a band corresponding to the expression of a truncated TBK1 protein missing amino acids 690-713. ß-Actin is shown as a loading control. Molecular weights are indicated in kDa (All TBK1- and Actin-bands in (d) result from the same Western blot but were re-arranged for better presentation).

Source data

Supplementary Figure 5 Uncropped IB images corresponding to Supplementary Figure 4 and 5

Supplementary Figure 6 Functional analysis of TBK1 missense variance

(a) Co-immunoprecipitation (Co-IP): Flag-TBK1-WT or the indicated mutants were expressed in HEK293T cells. The fusion proteins were purified and immobilized from HEK293T cell lysates using anti-Flag beads followed by incubation with recombinant GST-OPTN. Co-precipitated GST-OPTN was detected with an antibody against OPTN (top panel). Protein levels of immobilized Flag-TBK1 WT were analyzed by immunoblotting (anti-Flag; lower panel). Flag-beads only (IgG only) were used as a negative control. WT (wild-type); KD (kinase-dead TBK1 mutation; p.Lys38Ala). (b) GST pull-down assay (upper panel): Flag-TBK1-WT or the indicated mutants from HEK293T cell lysates (as in a) were incubated with recombinant, immobilized GST-OPTN. Equal GST-OPTN protein levels were confirmed by Ponceau S staining. Arrowhead points to full-length GST-OPTN. Flag-TBK1 protein expression was analyzed by immunoblotting (lower panel). (c) Co-IP of myc-IRF3 and Flag-TBK1-WT or the indicated mutants from HEK293T cells using anti-Flag agarose. (Co-)Immunoprecipitated proteins were analyzed by IB with the indicated antibodies (upper panel). Asterisk points to heavy IgG bands. Flag-TBK1 and myc-IRF3 protein expression was analyzed by IB with the indicated antibodies (lower panel). IB images are representatives of three repetitions.

Supplementary Figure 7 Rare ALS-associated missense variants identified in our study (MAF < 0.0001) mapped onto the 3D structure* of human TBK1.

Shown is a TBK1 dimer, in which one monomer is white, and the second one is coloured according to domain structure (kinase domain, blue; ubiquitin-like domain, orange; coiled-coil domain 1, green). The N and C termini of both monomers are labelled, the additional ends of the protein chain relate to missing loops in the crystal structure. Displayed variants are p.Arg47His (R47H), p.Tyr105Ser (Y105S), p.Ile305Thr (I305T), p.Arg308Gln (R308Q), p.Arg357Gln (R357Q), p.Met559Arg (M559R), p.Ala571Val (A571V), p.Met598Val (M598V) and p.Glu643del (E643del). The variants selected for functional studies based on 3D structure modeling are highlighted in red. Met559 is buried inside a bundle of 3 helices, and the p.Met559Arg substitution must induce at least a local structural rearrangement, which could lead to effects on overall folding and dimerization. Arg308 is at the interface between the kinase and ubiquitin-like domains, and the p.Arg308Gln mutation will break its salt bridge to Asp304. Arg357 is located within the ubiquitin-like domain, but also at the dimer interface, and it is heavily involved in a salt bridge network. The p.Arg357Gln substitution must affect this network of interactions central to TBK1 dimerization. The locations of the C termini are indicated; note that the very C-terminal domain after residue 657 (containing the p.Glu696Lys mutation) was not included in the crystal structure. The p.Glu696Lys mutation was nevertheless included in the functional analysis due to its position in the functionally important coiled-coil domain, as was also the p.Arg47His variant because of its location in the kinase domain and its high evolutionary conservation. * Larabi, A. et al. Crystal structure and mechanism of activation of TANK-binding kinase 1. Cell Rep 3, 734–46 (2013).

Supplementary Figure 8 Uncropped IB images corresponding to Figure 3

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1–8 and Supplementary Tables 1, 4–9 (PDF 10421 kb)

Supplementary Methods Checklist

Reporting Checklist for Nature Neuroscience (PDF 114 kb)

Supplementary Table 2

List of TBK1 variants identified in the present study and available by the Exome Aggregation Consortium (ExAC). (XLSX 61 kb)

Supplementary Table 3

Clinical characteristics of TBK1 mutation carriers. (XLSX 16 kb)

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Freischmidt, A., Wieland, T., Richter, B. et al. Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia. Nat Neurosci 18, 631–636 (2015). https://doi.org/10.1038/nn.4000

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