Somatic TARDBP variants as a cause of semantic dementia

Abstract

The aetiology of late-onset neurodegenerative diseases is largely unknown. Here we investigated whether de novo somatic variants for semantic dementia can be detected, thereby arguing for a more general role of somatic variants in neurodegenerative disease. Semantic dementia is characterized by a non-familial occurrence, early onset (<65 years), focal temporal atrophy and TDP-43 pathology. To test whether somatic variants in neural progenitor cells during brain development might lead to semantic dementia, we compared deep exome sequencing data of DNA derived from brain and blood of 16 semantic dementia cases. Somatic variants observed in brain tissue and absent in blood were validated using amplicon sequencing and digital PCR. We identified two variants in exon one of the TARDBP gene (L41F and R42H) at low level (1-3%) in cortical regions and in dentate gyrus in two semantic dementia brains, respectively. The pathogenicity of both variants is supported by demonstrating impaired splicing regulation of TDP-43 and by altered subcellular localization of the mutant TDP-43 protein. These findings indicate that somatic variants may cause semantic dementia as a non-hereditary neurodegenerative disease, which might be exemplary for other late-onset neurodegenerative disorders.

Keywords: TARDBP; TDP-43; semantic dementia; somatic variants.

Figure 1

Flowchart of data filtering and analysis. From top left: Raw somatic variant calling using blood and dentate gyrus (DG) or medial temporal gyrus (MTG) deep exome sequencing data (WES), signal to noise filter (S2N), minor allele frequency filter (MAF), CADD score filter, annotating and grouping per gene, resulting in the genes affected in each patient with semantic dementia (SD). Right: Grouping genes affecting multiple patients (>5) or affecting candidate genes in fewer patients (one to four) to be included in the validation amplicon panel. To excluded false negative findings in the WES data in FTD-TDP known germline causal genes GRN and TARDBP, all exons in these genes were included in the validation panel. The first validation round was performed on the same tissues as the discovery WES to confirm true positive variants from the WES, or identify false negative findings in GRN or TARDBP. The second round of validation further classified true positive variants in additional brain tissues and non-demented controls.

Figure 2

Allele frequencies for L41F and R42H in all tested amplicon panel samples. Each column is a sample, the tissues represented by colour; blood (BL, blue), cerebellum (CER, green), dentate gyrus (DG, red), hippocampus (HIP, orange), middle temporal gyrus (MTG, purple), middle frontal gyrus (MFG, salmon) and superior parietal lobe (LPS, pink). The vertical axis shows the variant allele frequency in that respective tissue, with lines representing the 0.25% and 0.50% thresholds. The tissues with highest variant allele frequency are labelled with the patient identifier and respective tissue.

Figure 3

Allelic discrimination plots of the digital droplet PCR for the R42H TARDBP somatic variant. Each marker represents a single droplet and its respective wild-type (horizontal axis) and variant (vertical axis) signal intensity. Five different tissues of the carrier were tested: blood, middle temporal gyrus (MTG), middle frontal gyrus (MFG), lateral parietal lobe (LPS), cerebellum (CER) and a negative control of water is shown. The grey droplets are considered empty, green droplets are wild-type only, orange is both wild-type and variant alleles, and blue droplets were harbouring only the variant allele.

Figure 4

Axial T₁-weighted MRI of the semantic dementia patient carrying somatic variant R42H, showing profound leftsided temporal atrophy 3 years after disease onset. Pathological examination 15 years after disease onset showed atrophy of both temporal poles. The middle image is from a patient without a somatic variant (4 years after onset) showing atrophy of both temporal lobes. Right: A patient with the germline (p.I383V) TARDBP variant, showing a similar atrophy pattern bilaterally (4 years after onset).

Figure 5

Impact of both TARDBP variants on the splicing regulation functionality of TDP-43, demonstrated by splice-in/out add-back assay of CFTR exon 9. From left to right: The first two lanes show the baseline measurement with both splicing in and out of exon 9 in the absence (-) or presence of TDP-43 siRNA (+). Lane 3 shows that addition of si-resistant wild-type TDP-43 can rescue the splicing functionality (WT) but this cannot be achieved by a TDP-43 carrying the F4L mutation that does not allow the protein to bind RNA (lane 4). Lanes 5 and 6 show the results obtained after the addition of mutated TDP-43 carrying the predicted damaging variants (R42H and L41F). Middle: Western blots showing equal expression of the flagged-TDP-43 wild-type and mutants (pFlag-TDP-43s) following knockdown of the endogenous protein (end. TDP-43). Tubulin was used as an internal control. Bottom: Quantification of the ratio of CFTR exon 9 inclusion. The standard deviation and P-values are reported for three independent experiments. Unpaired t-test was performed for statistical analysis (*P < 0.05).

Figure 6

Impact of TARDBP variants on the localization of flagged-TDP-43 wild-type and mutant proteins overexpressed in HeLa cells. The overexpressed proteins were visualized using anti-Flag polyclonal antibody in a 100 nm/pixel field. Scale bar = 10 nm. Top row: Wild-type Flag TDP-43, followed by Flagged TDP-43s carrying both variants; L41F and R42H. Left column: DAPI staining to indicate the chromatin in the nucleus in blue. Middle column: TDP-43 stained in red with a Flag-specific antibody. Right column: Merged images demonstrating TDP-43 localization in the nucleus for wild-type TDP-43, whilst localizing also in the cytoplasm for both TDP43 with variant R42H and L41F. Bottom: Box plots showing fluorescent TDP-43 signal is quantified in the nucleus and cytoplasm for nine cells of each line. The average ratio of nuclear and cytosolic signal is plotted and compared between groups. ****P < 0.0001 and ***P < 0.001 as calculated by two-way ANOVAs between the groups illustrated.