TBK1 mutations in Italian patients with amyotrophic lateral sclerosis: genetic and functional characterisation

L Pozzi, F Valenza, Lorena Mosca, Andrea Dal Mas, Teuta Domi, A Romano, C Tarlarini, YM Falzone, L Tremolizzo, G Sorarù, F Cerri, Pilar M. Ferraro, S Basaia, F Agosta, R Fazio, M Comola, G Comi, M Ferrari, A Quattrini, C LunettaS Penco, D Bonanomi, P Carrera, N Riva

Research output: Contribution to journalArticle

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. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
Original languageEnglish
Pages (from-to)869-875
Number of pages7
JournalJournal of neurology, neurosurgery, and psychiatry
Volume88
Issue number10
DOIs
Publication statusPublished - 2017

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Amyotrophic Lateral Sclerosis
Phosphotransferases
Mutation
Fibroblasts
Phosphorylation
Genes
Haploinsufficiency
RNA Precursors
Protein Kinases
Plasmids

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TBK1 mutations in Italian patients with amyotrophic lateral sclerosis: genetic and functional characterisation. / Pozzi, L; Valenza, F; Mosca, Lorena; Dal Mas, Andrea; Domi, Teuta; Romano, A; Tarlarini, C; Falzone, YM; Tremolizzo, L; Sorarù, G; Cerri, F; Ferraro, Pilar M.; Basaia, S; Agosta, F; Fazio, R; Comola, M; Comi, G; Ferrari, M; Quattrini, A; Lunetta, C; Penco, S; Bonanomi, D; Carrera, P; Riva, N.

In: Journal of neurology, neurosurgery, and psychiatry, Vol. 88, No. 10, 2017, p. 869-875.

Research output: Contribution to journalArticle

Pozzi, L, Valenza, F, Mosca, L, Dal Mas, A, Domi, T, Romano, A, Tarlarini, C, Falzone, YM, Tremolizzo, L, Sorarù, G, Cerri, F, Ferraro, PM, Basaia, S, Agosta, F, Fazio, R, Comola, M, Comi, G, Ferrari, M, Quattrini, A, Lunetta, C, Penco, S, Bonanomi, D, Carrera, P & Riva, N 2017, 'TBK1 mutations in Italian patients with amyotrophic lateral sclerosis: genetic and functional characterisation', Journal of neurology, neurosurgery, and psychiatry, vol. 88, no. 10, pp. 869-875. https://doi.org/10.1136/jnnp-2017-316174
Pozzi, L ; Valenza, F ; Mosca, Lorena ; Dal Mas, Andrea ; Domi, Teuta ; Romano, A ; Tarlarini, C ; Falzone, YM ; Tremolizzo, L ; Sorarù, G ; Cerri, F ; Ferraro, Pilar M. ; Basaia, S ; Agosta, F ; Fazio, R ; Comola, M ; Comi, G ; Ferrari, M ; Quattrini, A ; Lunetta, C ; Penco, S ; Bonanomi, D ; Carrera, P ; Riva, N. / TBK1 mutations in Italian patients with amyotrophic lateral sclerosis: genetic and functional characterisation. In: Journal of neurology, neurosurgery, and psychiatry. 2017 ; Vol. 88, No. 10. pp. 869-875.
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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. {\circledC} Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.",
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T1 - TBK1 mutations in Italian patients with amyotrophic lateral sclerosis: genetic and functional characterisation

AU - Pozzi, L

AU - Valenza, F

AU - Mosca, Lorena

AU - Dal Mas, Andrea

AU - Domi, Teuta

AU - Romano, A

AU - Tarlarini, C

AU - Falzone, YM

AU - Tremolizzo, L

AU - Sorarù, G

AU - Cerri, F

AU - Ferraro, Pilar M.

AU - Basaia, S

AU - Agosta, F

AU - Fazio, R

AU - Comola, M

AU - Comi, G

AU - Ferrari, M

AU - Quattrini, A

AU - Lunetta, C

AU - Penco, S

AU - Bonanomi, D

AU - Carrera, P

AU - Riva, N

PY - 2017

Y1 - 2017

N2 - 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. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

AB - 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. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

U2 - 10.1136/jnnp-2017-316174

DO - 10.1136/jnnp-2017-316174

M3 - Article

VL - 88

SP - 869

EP - 875

JO - Journal of Neurology, Neurosurgery and Psychiatry

JF - Journal of Neurology, Neurosurgery and Psychiatry

SN - 0022-3050

IS - 10

ER -