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

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.

SUBMITTER: Pozzi L

PROVIDER: S-EPMC5629935 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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<i>TBK1</i> mutations in Italian patients with amyotrophic lateral sclerosis: genetic and functional characterisation.

Pozzi Laura L Valenza Fabiola F Mosca Lorena L Dal Mas Andrea A Domi Teuta T Romano Alessandro A Tarlarini Claudia C Falzone Yuri Matteo YM Tremolizzo Lucio L Sorarù Gianni G Cerri Federica F Ferraro Pilar M PM Basaia Silvia S Agosta Federica F Fazio Raffaella R Comola Mauro M Comi Giancarlo G Ferrari Maurizio M Quattrini Angelo A Lunetta Christian C Penco Silvana S Bonanomi Dario D Carrera Paola P Riva Nilo N

Journal of neurology, neurosurgery, and psychiatry 20170819 10

<h4>Background</h4><i>TANK-binding kinase 1</i> (<i>TBK1</i>) gene has been recently identified as a causative gene of amyotrophic lateral sclerosis (ALS).<h4>Methods</h4>We sequenced the <i>TBK1</i> gene in a cohort of 154 Italian patients with ALS with unclear genetic aetiology. We subsequently assessed the pathogenic potential of novel identified <i>TBK1</i> variants using functional in vitro studies: expression, targeting and activity were evaluated in patient-derived fibroblasts and in cell ...[more]

PMID: 28822984

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Project description:BackgroundAmyotrophic lateral sclerosis (ALS) is a complex, late-onset, neurodegenerative disease with a genetic contribution to disease liability. Genome-wide association studies (GWAS) have identified ten risk loci to date, including the TNIP1/GPX3 locus on chromosome five. Given association analysis data alone cannot determine the most plausible risk gene for this locus, we undertook a comprehensive suite of in silico, in vivo and in vitro studies to address this.MethodsThe Functional Mapping and Annotation (FUMA) pipeline and five tools (conditional and joint analysis (GCTA-COJO), Stratified Linkage Disequilibrium Score Regression (S-LDSC), Polygenic Priority Scoring (PoPS), Summary-based Mendelian Randomisation (SMR-HEIDI) and transcriptome-wide association study (TWAS) analyses) were used to perform bioinformatic integration of GWAS data (Ncases = 20,806, Ncontrols = 59,804) with 'omics reference datasets including the blood (eQTLgen consortium N = 31,684) and brain (N = 2581). This was followed up by specific expression studies in ALS case-control cohorts (microarray Ntotal = 942, protein Ntotal = 300) and gene knockdown (KD) studies of human neuronal iPSC cells and zebrafish-morpholinos (MO).ResultsSMR analyses implicated both TNIP1 and GPX3 (p < 1.15 × 10-6), but there was no simple SNP/expression relationship. Integrating multiple datasets using PoPS supported GPX3 but not TNIP1. In vivo expression analyses from blood in ALS cases identified that lower GPX3 expression correlated with a more progressed disease (ALS functional rating score, p = 5.5 × 10-3, adjusted R2 = 0.042, Beffect = 27.4 ± 13.3 ng/ml/ALSFRS unit) with microarray and protein data suggesting lower expression with risk allele (recessive model p = 0.06, p = 0.02 respectively). Validation in vivo indicated gpx3 KD caused significant motor deficits in zebrafish-MO (mean difference vs. control ± 95% CI, vs. control, swim distance = 112 ± 28 mm, time = 1.29 ± 0.59 s, speed = 32.0 ± 2.53 mm/s, respectively, p for all < 0.0001), which were rescued with gpx3 expression, with no phenotype identified with tnip1 KD or gpx3 overexpression.ConclusionsThese results support GPX3 as a lead ALS risk gene in this locus, with more data needed to confirm/reject a role for TNIP1. This has implications for understanding disease mechanisms (GPX3 acts in the same pathway as SOD1, a well-established ALS-associated gene) and identifying new therapeutic approaches. Few previous examples of in-depth investigations of risk loci in ALS exist and a similar approach could be applied to investigate future expected GWAS findings.

Dataset Information

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

Publications

<i>TBK1</i> mutations in Italian patients with amyotrophic lateral sclerosis: genetic and functional characterisation.

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