TBK1 Mutation Spectrum in an Extended European Patient Cohort with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis

Abstract

We investigated the mutation spectrum of the TANK-Binding Kinase 1 (TBK1) gene and its associated phenotypic spectrum by exonic resequencing of TBK1 in a cohort of 2,538 patients with frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), or FTD plus ALS, ascertained within the European Early-Onset Dementia Consortium. We assessed pathogenicity of predicted protein-truncating mutations by measuring loss of RNA expression. Functional effect of in-frame amino acid deletions and missense mutations was further explored in vivo on protein level and in vitro by an NFκB-induced luciferase reporter assay and measuring phosphorylated TBK1. The protein-truncating mutations led to the loss of transcript through nonsense-mediated mRNA decay. For the in-frame amino acid deletions, we demonstrated loss of TBK1 or phosphorylated TBK1 protein. An important fraction of the missense mutations compromised NFκB activation indicating that at least some functions of TBK1 are lost. Although missense mutations were also present in controls, over three times more mutations affecting TBK1 functioning were found in the mutation fraction observed in patients only, suggesting high-risk alleles (P = 0.03). Total mutation frequency for confirmed TBK1 LoF mutations in the European cohort was 0.7%, with frequencies in the clinical subgroups of 0.4% in FTD, 1.3% in ALS, and 3.6% in FTD-ALS.

Keywords: ALS; FTD; NFκB luciferase reporter assay; TANK-Binding Kinase 1; TBK1; amyotrophic lateral sclerosis; frontotemporal dementia; mutations.

Figure 1

Transcript and protein analysis of TBK1 LoF and single amino acid deletion mutations. A: gDNA and cDNA sequence traces around the c.288delT (p. Val97Phefs^*2) mutation, showing reduced expression of the mutant transcript on cDNA extracted from blood. B: gDNA and cDNA sequence traces around the c.379C>T (p.Arg127^*) mutation, showing the absence of the mutant transcript on cDNA extracted from blood. C: Sizing of cDNA fragments generated with primers in TBK1 exon 10 and exon 13 of the c.1340+1G>A (p.Ala417^*) carrier on cDNA extracted from blood. Sequence traces from the low‐expressed aberrant transcript demonstrates skipping of exon 11. D: Transcript and protein analysis on brain frontal cortex from the c.235_237delACA (p.Thr79del) carrier and four age‐matched control brains. The graph on the left shows the relative expression in the patient sample (blue) compared with the control samples (black) measured by quantitative real‐time PCR (qRT‐PCR). In the middle, Western blot analysis is shown of protein extracts from the patient carrier compared with control individuals. The upper band represents TBK1 (84 kDa) and the lower band represents the housekeeping protein GAPDH (37 kDa). The graph on the right shows the quantification in the patient sample (blue) and control samples (black) of the TBK1 signal normalized to the signal of GAPDH. Error bars represent the SD. E: Western blot analysis of phosphorylated TBK1 (Ser172, p‐TBK1) (upper band, 84 kDa) in HEK293T cells overexpressing the in‐frame single amino acid deletions (p.Thr79del, p.Asp167del, and p.Glu643del) compared with wild type, relative to GAPDH (lower band, 37 kDa). Mock and kinase dead (p.Ser172Ala, KD) were used as negative control. cDNA numbering according to reference sequence NM_013254.3, in addition, for intronic variants, the genomic reference sequence NC_000012.12 was used. Nucleotide positions refer to cDNA sequence and nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1. Protein numbering according to reference sequence NP_037386.1.

Figure 2

Impact of mutant TBK1 on NFκB activity in the IFN pathway. Graphical representation of the mean NFκB‐induced luciferase activity of identified in‐frame amino acid deletions and missense mutations found in patients‐only, shared by patients and controls, and in controls‐only, normalized to the mean signal from wild type. Luciferase activities were measured in at least three independent experiments and measured five times per experiment. The different domains are indicated in different colors as shown in the figure legend. WT, wild type TBK1 vector; Mock, empty vector containing no TBK1; S172A‐KD, p.Ser172Ala TBK1 kinase dead mutation. Mock and S172A‐KD were used as negative controls. Error bars depict standard deviation and asterisks above the bars indicate significant difference from the wild‐type level after Bonferroni correction (P<0.001). Protein numbering according to reference sequence NP_037386.1.

Figure 3

Neuropathology features of the FTD‐ALS patient with the TBK1 p.Thr79del mutation. Severe neuronal loss, gliosis, and loosening of the neuropil are observed in entorhinal and transentorhinal region (lower image) as compared with relatively well‐preserved frontal cortex (upper image) (HE) (A). Abundant TDP‐43 protein aggregates in neurons and oligodendroglial cells (B), better seen in (C) at higher magnification (arrows). Hippocampal dentate gyrus shows some granular neurons lacking physiological nuclear immunoreactivity but shifting toward pathological inclusions in the cytoplasm (D). Morphological spectrum of neuronal cytoplasmic inclusions (E–J), seen as compact bodies (E), diffuse granular cytoplasmic immunoreactivity or “preinclusion” type (F), skein‐like inclusions (G, inset), compact ring‐like inclusions (H), or a combination of diffuse cytoplasmic and compact in the same motor neuron (I). Signs of corticospinal tract degeneration at the level of the spinal cord with marked reduction of axonal density as shown by antineurofilament immunohistochemistry (K, inset shows regular density of axons for comparison), and increased macrophagic activity (L, anti‐CD68 immunohistochemistry, inset shows regular density of CD68+ cells in the spinal cord for comparison). Neuropathological features of concomitant argyrophilic grain pathology (M–P). Ballooned cells are seen in amygdala (M) and are nicely stained by hyperphosphorylated tau (AT8, inset). Moreover, frequent hpTau‐positive grains, mainly composed of four‐repeat tau isoforms, are detected in the limbic system (N, CA1 sector is shown), and represent enlargements/verrucosities of dendritic spines (N, inset). Oligodendroglial coiled bodies (O) and bush‐like astrocytes (P) accompany the full picture.