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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.

Project description:Abstract An expansion of the GGGGCC hexanucleotide in the non-coding region of C9orf72 represents the most common cause of familial amyotrophic lateral sclerosis. The objective was to describe and analyse the clinical and genetic features of amyotrophic lateral sclerosis patients with C9orf72 mutations in a large population. Between November 2011 and December 2020, clinical and genetic characteristics of n = 248 patients with amyotrophic lateral sclerosis carrying C9orf72 mutations were collected from the clinical and scientific network of German motoneuron disease centres. Clinical parameters included age of onset, diagnostic delay, family history, neuropsychological examination, progression rate, phosphorylated neurofilament heavy chain levels in CSF and survival. The number of repeats was correlated with the clinical phenotype. The clinical phenotype was compared to n = 84 patients with SOD1 mutations and n = 2178 sporadic patients without any known disease-related mutations. Patients with C9orf72 featured an almost balanced sex ratio with 48.4% (n = 120) women and 51.6% (n = 128) men. The rate of 33.9% patients (n = 63) with bulbar onset was significantly higher compared to sporadic (23.4%, P = 0.002) and SOD1 patients (3.1%, P < 0.001). Of note, 56.3% (n = 138) of C9orf72, but only 16.1% of SOD1 patients reported a negative family history (P < 0.001). The GGGGCC hexanucleotide repeat length did not influence the clinical phenotypes. Age of onset (58.0, interquartile range 52.0–63.8) was later compared to SOD1 (50.0, interquartile range 41.0–58.0; P < 0.001), but earlier compared to sporadic patients (61.0, interquartile range 52.0–69.0; P = 0.01). Median survival was shorter (38.0 months) compared to SOD1 (198.0 months, hazard ratio 1.97, 95% confidence interval 1.34–2.88; P < 0.001) and sporadic patients (76.0 months, hazard ratio 2.34, 95% confidence interval 1.64–3.34; P < 0.001). Phosphorylated neurofilament heavy chain levels in CSF (2880, interquartile range 1632–4638 pg/ml) were higher compared to sporadic patients (1382, interquartile range 458–2839 pg/ml; P < 0.001). In neuropsychological screening, C9orf72 patients displayed abnormal results in memory, verbal fluency and executive functions, showing generally worse performances compared to SOD1 and sporadic patients and a higher share with suspected frontotemporal dementia. In summary, clinical features of patients with C9orf72 mutations differ significantly from SOD1 and sporadic patients. Specifically, they feature a more frequent bulbar onset, a higher share of female patients and shorter survival. Interestingly, we found a high proportion of patients with negative family history and no evidence of a relationship between repeat lengths and disease severity. Wiesenfarth et al. report that amyotrophic lateral sclerosis patients with C9orf72 mutations differ significantly from sporadic patients and SOD1 gene carriers, including a higher share of bulbar onset, female patients, more severe neuropsychological deficits and shorter survival. No evidence of a relationship between repeat lengths and disease severity was found. Graphical Abstract Graphical Abstract

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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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