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Neurogenetics
Research paper
TBK1 mutations in Italian patients with amyotrophic lateral sclerosis: genetic and functional characterisation
  1. Laura Pozzi1,
  2. Fabiola Valenza2,
  3. Lorena Mosca3,
  4. Andrea Dal Mas2,
  5. Teuta Domi1,
  6. Alessandro Romano1,
  7. Claudia Tarlarini3,
  8. Yuri Matteo Falzone1,4,
  9. Lucio Tremolizzo5,
  10. Gianni Sorarù6,
  11. Federica Cerri1,4,
  12. Pilar M Ferraro7,
  13. Silvia Basaia7,
  14. Federica Agosta7,
  15. Raffaella Fazio4,
  16. Mauro Comola4,
  17. Giancarlo Comi4,8,
  18. Maurizio Ferrari8,9,
  19. Angelo Quattrini1,
  20. Christian Lunetta10,
  21. Silvana Penco3,
  22. Dario Bonanomi2,
  23. Paola Carrera9,
  24. Nilo Riva1,4
  1. 1 Neuropathology Unit, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy
  2. 2 Molecular Neurobiology Laboratory, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy
  3. 3 Medical Genetic Unit, Department of Laboratory Medicine, Niguarda Hospital, Milan, Italy
  4. 4 Department of Neurology, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy
  5. 5 Neurology Unit, “San Gerardo” Hospital and University of Milano-Bicocca, Monza, Italy
  6. 6 Department of Neurosciences, Neuromuscular Center, University of Padova, Padua, Italy
  7. 7 Neuroimaging Research Unit, Department of Neurology, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy
  8. 8 Università Vita e Salute San Raffaele, Milan, Italy
  9. 9 Division of Genetics and Cell Biology, Unit of Genomics for Human Disease Diagnosis, San Raffaele Scientific Institute, Milan, Italy
  10. 10 NEuroMuscular Omnicentre (NEMO), Fondazione Serena Onlus, Milan, Italy
  1. Correspondence to Dr Nilo Riva, Department of Neurology, INSPE and Division of Neuroscience, San Raffaele Scientific Institute, via Olgettina 48, 20132 Milan, Italy; riva.nilo{at}hsr.it

Abstract

Background TANK-binding kinase 1 (TBK1) gene has been recently identified as a causative gene of amyotrophic lateral sclerosis (ALS).

Methods We sequenced the TBK1 gene in a cohort of 154 Italian patients with ALS with unclear genetic aetiology. We subsequently assessed the pathogenic potential of novel identified TBK1 variants using functional in vitro studies: expression, targeting and activity were evaluated in patient-derived fibroblasts and in cells transfected with mutated-TBK1 plasmids.

Results We identified novel genomic TBK1 variants including two loss-of-function (LoF) (p.Leu59Phefs*16 and c.358+5G>A), two missense (p.Asp118Asn and p.Ile397Thr) and one intronic variant (c.1644–5_1644-2delAATA), in addition to two previously reported pathogenetic missense variants (p.Lys291Glu and p.Arg357Gln). Functional studies in patient-derived fibroblasts revealed that the c.358+5G>A causes aberrant pre-mRNA processing leading TBK1 haploinsufficiency. Biochemical studies in cellular models showed that the truncating variant p.Leu59Phefs*16 abolishes TBK1 protein expression, whereas the p.Asp118Asn variant severely impairs TBK1 phosphorylation activity. Conversely, the p.Ile397Thr variant displayed enhanced phosphorylation activity, whose biological relevance is not clear.

Conclusion The observed frequency of TBK1 LoF variants was 1.3% (2/154), increasing up to 3.2% (5/154) by taking into account also the functional missense variants that we were able to classify as potentially pathogenic, supporting the relevance of TBK1 in the Italian population with ALS.

This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/

https://doi.org/10.1136/jnnp-2017-316174

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    Footnotes

    • Contributors LP, FV, LM, ADM, TD, AR and CT performed the experiments and acquired the data; LP, FV, LM, ADM, TD, AR, FA, SP, DB, PC and NR contributed to analysis and interpretation of the data; YMF, LT, GS, FC, RF, FA, CL and NR recruited the patients, performed the clinical evaluation, acquired clinical data and collected patient DNA; LP, FV, ADM, AR, YMF, DB, PC and NR drafted and revised the manuscript; LT, GS, FA, GC, MF, AQ, CL, SP, DB, PC and NR critically revised the manuscript for intellectual content; AQ, CL, SP, DB, PC and NR conceptualised and designed the study, and interpreted the analysis; FA, GC, MF, AQ, CL, SP, DB, PC and NR obtained the funding; AQ, SP, DB, PC and NR supervised the study; all authors had full access to the data in the study and approved the final version of the manuscript.

    • Funding This work was supported by AriSLA, Italian Research Foundation for ALS (ExAlta Grant) to AQ, the Giovanni Armenise-Harvard Foundation and ERC-StG 35590–NEVAI to DB and the Italian Ministry of Health (grant RF-2010-2313220) to AF.

    • Competing interests None declared.

    • Patient consent Obtained.

    • Ethics approval The study was approved by the local Ethical Committee of the San Raffaele Scientific Institute.

    • Provenance and peer review Not commissioned; externally peer reviewed.