Mutations in the 3′ untranslated region of FUS causing FUS overexpression are associated with amyotrophic lateral sclerosis

Abstract

Mutations in the gene encoding fused-in-sarcoma (FUS) have been identified in a subset of patients with sporadic and familial amyotrophic lateral sclerosis (ALS). Variants in the 3' untranslated region (3'UTR) of FUS have also been reported in ALS patients, but their pathogenic role has not been assessed. We sequenced the whole 3'UTR of FUS in 420 ALS patients who were negative for mutations in the currently known ALS genes and in 480 ethnically matched controls. We detected four 3'UTR variants (c.*48 G>A, c.*59 G>A, c.*108 C>T and c.*110 G>A) in four sporadic and in one familial ALS patients compared with none in controls (P = 0.02).We investigated whether these variants impaired FUS expression in primary fibroblast cultures from three patients harbouring the c.*59 G>A, c.*108 C>T and c.*110 G>A variants, respectively. The pattern of FUS expression was also investigated in fibroblasts from one ALS patient with FUS R521C mutation, in two ALS patients without mutations in the known ALS genes and in four control individuals. By immunostaining and immunoblotting, large amounts of FUS were observed in both the cytoplasm and nuclei of mutant 3'UTR FUS fibroblasts. In FUS R521C mutant fibroblasts, we observed a slight increase of FUS in the cytoplasm associated with a remarkable loss of detection in nuclei. Our findings show that mutations in 3'UTR of FUS are overrepresented in ALS patients and result into translation de-regulation of FUS. Overexpression and mislocalization of wild-type FUS likely contribute to ALS pathogenesis in these cases.

Activity-dependent FUS dysregulation disrupts synaptic homeostasis

“…The CAG-FUS WT and CAG-FUS R521G mouse models demonstrate that increased expression of FUS alone can cause cellular toxicity. This result is consistent with the recent finding that mutations in the 3′ UTR of FUS increase FUS expression levels and cause ALS (15). This result is also in agreement with end-stage pathological markers or outcomes in human patients.…”

“…Besides missense mutations at the C terminus of FUS protein, overexpression of wild-type FUS caused by mutations in its 3′ UTR also has been linked to ALS (15), suggesting that overexpression of wild-type FUS is pathogenic under certain circumstances. Indeed, pathogenic effects of increased levels of wild-type proteins are common in other neurodegenerative disorders, as exemplified by increased gene dose or overexpression of wild-type TDP-43, α-synuclein, and amyloid β precursor protein (APP) in ALS/FTLD, Parkinson disease, and Alzheimer's disease (16)(17)(18).…”

An ALS-associated mutation in the FUS 3′-UTR disrupts a microRNA–FUS regulatory circuitry

“…Nevertheless, the increase in FUS protein did not reach the levels observed with the entire deletion of the 3 0 -UTR (FUS-D3 0 -UTR), suggesting the presence of additional regulatory elements besides the MRE (miRNA recognition element) for miR-141/200a. This seems indeed to be the case since three other mutations, associated with severe ALS phenotypes and linked to high accumulation of protein, were also identified in this region 7 . However, according to TargetScan and PicTar analyses 12,13 , none of those mutations appear to affect conserved miRNA-binding sites.…”

“…Bioinformatics search 12,13 for miRNA-responsive elements in the FUS 3 0 -UTR indicated that among the identified 3 0 -UTR mutations 7 , two patients carried the G48A substitution (in one case of inherited type), which localizes to a predicted binding site for miR-141 and miR-200a. These two miRNAs share the same seed sequence and belong to the same miRNA family 14 The members of this family are of particular interest for human health and disease, because they have been shown to be downregulated during tumour progression and to act as key regulators of epithelial-to-mesenchymal transition 15 .…”

“…Most of these mutations alter the subcellular partitioning of the protein, which leads to a decrease of the nuclear content and an increase in the cytoplasm. Notably, recent sequencing of the 3 0 -untranslated region (3 0 -UTR) of FUS messenger RNA (mRNA) in 420 ALS patients, negative for mutations in the currently known ALS-associated genes, identified four mutations linked to severe outcomes that caused a strong increase in FUS accumulation 7 . The pathogenetic effects of wild-type (wt) FUS overexpression observed in these patients suggests that not only the nuclearcytoplasmic imbalance of the mutant protein, but also alteration of the physiological levels of the wt protein may contribute to ALS pathogenesis.…”