Identification of FUS p.R377W in essential tremor

Abstract

Background

Exome sequencing analysis has recently identified a nonsense mutation in fused in sarcoma (FUS) segregating with essential tremor (ET) within a large French-Canadian family. Further characterization of FUS resulted in the identification of additional mutations in ET patients; however their pathogenicity still remains to be confirmed. We set out to evaluate the role of FUS in an independent cohort of ET patients from Canada.

Methods

We analyzed the entire coding sequence of FUS in 217 patients diagnosed with ET and genotyped two missense variants in 219 healthy controls by Sanger sequencing.

Results

Sequencing of FUS identified a previously reported non-pathogenic mutation p.G174_G175del in one ET patient and two healthy controls, and a novel p.R377W in one patient with family history of disease. This mutation is highly conserved and strongly predicted to be damaging by in-silico analysis.

Conclusion

This study has identified a novel FUS p.R377W substitution in ET patients. Additional genotyping studies in large number of ET patients and controls are necessary to conclusively define its pathogenicity.

Introduction

Essential tremor (ET) is a highly prevalent neurological disorder affecting 4% of the population above age 40. It is clinically characterized by postural and kinetic tremor that worsens with movement.[1] Exome sequencing analysis in a French-Canadian family with ET identified a novel nonsense mutation (p.Q290*) in fused in sarcoma (FUS) segregating with disease. FUS, which has been previously implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is a DNA/RNA-binding nucleoprotein involved DNA repair, and the regulation of transcription, splicing, and transport of mRNA from the nucleus to the cytoplasm.[2] Screening of additional cases and controls identified two ET patients and one control with a p.R216C mutation, and one patient with a p.P431L mutation. The p.R216C mutation had been previously reported in one ALS case,[3] and in silico analysis predicts this mutation to be damaging; however, the identification of p.R216C in one healthy individual and the lack of additional family members for segregation analysis render the pathogenicity of this mutation questionable.[2] Similar confirmation of pathogenicity is required for p.P431L. Replication studies have so far failed to conclusively confirm a role for FUS in ET.[4–7] Given these findings, we set out to evaluate the role of FUS in an independent cohort of ET patients from Canada.

Methods

We analyzed 217 ET patients and 219 controls of European descent recruited at the Saskatoon Health Region Royal University Hospital. All patients diagnosed with disease satisfied clinical criteria for “definite” or “probable” ET.[8] The patient group presented a mean age of 72.1 ± 15.1 years and age at diagnosis of 54.4 ± 18.3 years. The control group consisted of unrelated individuals and spouses with an average age of 71.0 ± 12.3 years. The ethical review board of each institution approved the study and all participants provided informed consent. Sequencing FUS exons (NM_004960.3) was performed as previously described.[9] Primer sequences are available on request.

Results

Sequencing analysis of FUS in 217 ET patients identified one homozygous carrier of a previously described six nucleotide deletion (c.520_525delGGAGGT) resulting in the loss of two glycine residues (p.G174_G175del), a novel heterozygous c.1129C>T (p.R377W) mutation, and three previously reported synonymous variants (p.G49G, rs741810; p.Y97Y, rs1052352; and p.R522R, rs138901914). The position of p.G174_G175del is ambiguous and was arbitrarily assigned to the most 3′ position possible and is equivalent to previously reported deletions of two contiguous glycine residues between positions 168 and 175. Sequencing 219 controls for the exons containing p.G174_G175del and p.R377W resulted in the identification of two p.G174_G175del healthy heterozygous carriers (age 69 and 70). No additional carriers of the p.R377W mutation were identified.

Discussion

Pathogenic mutations in FUS have been reported in patients suffering from ALS, FTD and more recently nominated for ET.[2–4, 10] Although three different FUS mutations have been identified in ET, replication studies failed to provide additional support.[4–7] To further define the prevalence of FUS mutations in ET we screened its entire coding region in an independent series of ET patients from Canada. This analysis identified one patient with a homozygote deletion (p.G174_G175del) that was initially reported as pathogenic for ALS, but more recently described as non-deleterious.[3, 10] Genotyping this deletion in controls resulted in the identification of two heterozygous carriers, thus providing additional support for this variant as a benign polymorphism. In addition to this deletion, we also identified a novel heterozygous p.R377W mutation in one ET patient with onset of disease at 67 years of age. The initial symptom was hand tremor, followed by bilateral action and postural tremor which worsened over 19 years of disease course requiring medication. Although this patient had positive family history of disease (Figure 1A), with one brother diagnosed with parkinsonism, and one sister and one uncle with possible ET, no additional family members were available for study. Clinical information for the brother indicated development of tremor at 78 years of age, with aggressive behavior, visual hallucinations and dementia within five years of diagnosis.

Figure 1. FUS p.R377W pedigree and cross-species conservation.

A) Males are represented by squares, females by circles, symbols with a number represent the number of additional sibs. A diagonal line indicates deceased subjects, and the probands indicated with an arrow head. Black filled symbols are used to indicate individuals diagnosed with ET; grey, possible ET; and black dot, parkinsonism. B) Protein orthologs were aligned via ClustalW. Amino acid position for FUS p.R377W is highlighted in black. Protein orthologs with amino acid positions differing from those of the human sequence are indicated in gray. RefSeq accession numbers: Homo sapiens, NP_004951.1; Mus musculus, NP_631888.1; Sus scrofa, AEQ61462.1; Gallus gallus, NP_001001531.1; Xenopus laevis, AAH44319.1 Danio rerio, NP_957377.2.

The p.R377W mutation, which has not been identified in over 4,800 individuals of European descent characterized through The 1000 Genomes (http://www.1000genomes.org) and the NHLBI GO Exome Sequencing Project (http://evs.gs.washington.edu), is highly conserved (Figure 1B) and strongly predicted to be damaging by SIFT (http://sift.jcvi.org) and polyphen (http://genetics.bwh.harvard.edu/pph2). This mutation is located in the first of two FUS arginine-glycine-rich domains, in which mutations for ALS and FTD have not been described. This is consistent with the previously reported p.Q209* mutation for ET, which is located in the nuclear export signal of FUS; suggesting the possibility for different pathogenic mechanisms for these diseases.[2] Although segregation analyses of the p.R377W mutation were not possible, thus hindering the evidence of pathogenicity, its high level of conservation and the identification in one patient with family history of disease still provides support for a role of FUS mutations in ET. However, additional genotyping studies in large number of ET patients and controls are necessary to conclusively define the pathogenicity of p.R377W.