Exome sequencing identifies FUS mutations as a cause of essential tremor

Abstract

Essential tremor (ET) is a common neurodegenerative disorder that is characterized by a postural or motion tremor. Despite a strong genetic basis, a gene with rare pathogenic mutations that cause ET has not yet been reported. We used exome sequencing to implement a simple approach to control for misdiagnosis of ET, as well as phenocopies involving sporadic and senile ET cases. We studied a large ET-affected family and identified a FUS p.Gln290(∗) mutation as the cause of ET in this family. Further screening of 270 ET cases identified two additional rare missense FUS variants. Functional considerations suggest that the pathogenic effects of ET-specific FUS mutations are different from the effects observed when FUS is mutated in amyotrophic lateral sclerosis cases; we have shown that the ET FUS nonsense mutation is degraded by the nonsense-mediated-decay pathway, whereas amyotrophic lateral sclerosis FUS mutant transcripts are not.

Figure 1

A Schematic of ET Variants within FUS

Figure 2

Expression of FUS mRNA Carrying ET and ALS Mutations (A) Lymphoblastoid cells derived from five ET-affected individuals (individuals II:13, III:14, IV:6, IV:11, and IV:12 in Figure S1) who carry the FUS c.868C>T (p.Gln290^∗) mutation and from ALS-affected individuals expressing three different FUS mutations (c.1555C>T [p.Gln519^∗], c.1562G>A [p.Arg521His], and c.1542-2A>C) were treated with the protein-synthesis inhibitor puromycin (300 μg for 6 hr). After this, treated cells were harvested, and total RNA was prepared (Trizol extraction, Invitrogen) alongside the total RNA of untreated cells. Quantitative RT-PCR reactions were then performed with a Taqman probe specific to FUS (^∗∗∗p value = 6.47 × 10⁻⁵ [comparing the difference in mRNA expression in ET and ALS untreated cells]; ^∗∗p value = 0.03 [comparing the difference in mRNA expression in ET and ALS treated cells]). For each group of affected (ET and ALS) individuals, the average levels of expression are plotted. t tests were used for statistics, and error bars correspond to the standard error of the mean (SEM). (B) Standard nonquantitative RT-PCR reactions were prepared with the RNA used in (A) and primers in the flanking exons (or UTRs) of each mutation. Amplified cDNA was sequenced, and whereas the ALS mutations were seen independently of a puromycin treatment, the ET mutation could only be seen in puromycin-treated cells. (C) Quantitative allele-specific expression measurements of the same five FUS c.868C>T ET individuals from above were made with a set of Taqman custom-designed probes and primers for the wild-type (c.868C) and mutant (c.868T) alleles. After the puromycin treatment, the expression of the mutant allele became detectable. The specificity of the custom probe for the c.868C>T transcript is demonstrated in Figure S5. All expression levels were normalized with the human 18S ribosomal RNA (rRNA) gene and were calculated in comparison to the average level of expression of two healthy controls. t tests were used for statistics, and error bars correspond to SEM.