Abstract
TANK-binding kinase 1 (TBK1) has been identified as a causative gene of amyotrophic lateral sclerosis (ALS) in the Caucasian population in 2015. Here, we sequenced for TBK1 variants in a cohort of 15 familial ALS (fALS) and 275 sporadic ALS (sALS) of Chinese origin by targeted next-generation sequencing. We identified one likely benign missense variant (p. Ser398Pro), two missense variants of uncertain significance (p. Ile37Leu and p. Tyr677Asn), and two novel heterozygous variants in introns of TBK1, c.1522-3T > G and c.2066 + 4A > G. We performed splicing assays through minigene plasmids and RNA pull-down assay to determine that the two substitutions of nucleotides disrupted the binding of the important splicing regulator hnRNPA1 and promoted aberrant pre-mRNA splicing modes. The c.1522-3T > G variant promoted nearly 50.0% of abnormal transcripts (3 different types of insertions and deletions (indels) in junction of intron 13-exon 14) and the c.2066 + 4A > G variant inhibited about 75.0% inclusion of exon 19, both causing premature stop codon and producing TBK1 protein without CCD2. Immunofluorescence analysis showed that the expression of TBK1 with intronic variants was lower since less TBK1 distribution was observed in HEK293T cells. Both patients carrying TBK1 c.1522-3T > G and c.2066 + 4A > G variants developed a rapidly progressive ALS, with a survival of 31 and 10 months, respectively. The frequency of loss of function (LoF) variants in TBK1 was 0.73% in sALS in our cohort. We emphasize that intronic sequencing and pre-mRNA splicing analysis cannot be ignored to demonstrate the complex mutational spectrum and pathogenesis of ALS.
Highlights
Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease characterized by degeneration of upper and lower motor neurons (Chou and Norris, 1993)
Synthetic RNAs were labeled with biotin and incubated with nuclear extract from HeLa cells, the RNA-binding protein complexes were interacted with anti-hnRNPA1 antibody and separated by SDS-PAGE. (F,G) Immunofluorescence analysis of subcellular distribution of TANK-binding kinase 1 (TBK1) in HEK293T cells overexpressed by TBK1 cDNA carrying c.1522-3T or c.1522-3G (F) and TBK1 cDNA carrying c.2066 + 4A or c.2066 + 4G (G) was visualized by anti-FLAG antibody followed by anti-mouse Alexa Fluor 594-conjugated secondary antibody and DAPI staining
We identified five TBK1 variants by targeted next-generation sequencing in a Chinese cohort of 275 sporadic ALS (sALS) patients and 15 familial ALS (fALS) probands, including three missense variants (p.Ile37Leu, p.Ser398Pro, p.Tyr677Asn) and two novel intronic mutations (c.1522-3T > G, c.2066 + 4A > G)
Summary
Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease characterized by degeneration of upper and lower motor neurons (Chou and Norris, 1993). ALS-associated mutations in SOD1 are identified in 12–20% of fALS patients and 1–2% of sALS patients. SOD1 is the most common gene associated with ALS in Asian populations (Zou et al, 2017). FUS mutations could be found in 5% of fALS patients and 1% of sALS patients (Andersen and Al-Chalabi, 2011). A C9orf repeat expansion was the genetic cause in up to 35% of fALS patients and about 5% of sALS patients (Renton et al, 2011; Zou et al, 2017). Many new genes have been found as causative or highly associated with ALS, like TBK1, MATR3, CHCHD10, TUBA4A, NEK1, and C21orf (Chia et al, 2018)
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