A novel splice site FUS mutation in a familial ALS case: effects on protein expression

Abstract

Objective: To investigate the impact of a novel heterozygous FUS mutation in the acceptor splice site of intron 14 (c.1542 − 1 g > t) on protein expression in Peripheral Blood Mononuclear Cells (PBMC) from a familial ALS patient. Methods: PBMC were isolated for mRNA analysis (cDNA synthesis, sequencing and one-step RT-PCR), Western Immunoblot (WI), and Immunofluorescence (IF). Results: cDNA analysis revealed the skipping of exon 15 and a premature stop codon at c.228. RT-PCR showed reduced FUS mRNA by more than half compared to a healthy control (HC) and an ALS patient without genetic mutations (wtALS). In WI FUS band intensity in the proband was 30–50% compared to HC and wtALS. An antibody expected to detect only the wild-type protein did not reveal any reduction of FUS band intensity compared to the other antibodies. IF showed no difference among HC, wtALS, and the proband. Discussion: The reduction of FUS mRNA and protein in PBMC suggests the absence of the truncated protein, probably due to nonsense-mediated decay, leading to loss of function.

Keywords:

• Amyotrophic lateral sclerosis
• FUS
• splice site mutation
• loss of function

Ethical approval

The study was performed following the approval of the ethical committee “Comitato Etico Interaziendale Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino”, in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. We collected written informed consent to participate to the study and for publication.

Declaration of interest

Antonio Canosa, Annarosa Lomartire, Giovanni De Marco, Maurizio Grassano, Maura Brunetti, Umberto Manera, Rosario Vasta, Paolina Salamone, Giuseppe Fuda, Luca Sbaiz, Salvatore Gallone, Cristina Moglia: no competing interest. Andrea Calvo has received a research grant from Cytokinetics. Adriano Chiò serves on scientific advisory boards for Mitsubishi Tanabe, Roche, Biogen, Cytokinetics, and AveXis, and has received a research grant from Italfarmaco. The authors alone are responsible for the content and writing of this article.

Data availability statement

Data will be available upon request by interested researchers.

Additional information

Funding

This work was supported by the Italian Ministry of Health (Ministero della Salute, Ricerca Sanitaria Finalizzata, grant RF-2016-02362405), the Progetti di Rilevante Interesse Nazionale program of the Italian Ministry of Education, University and Research (grant 2017SNW5MB); and the Joint Programme–Neurodegenerative Disease Research (Strength, ALS-Care and Brain-Mend projects), granted by the Italian Ministry of Education, University and Research. This study was performed under the Department of Excellence grant of the Italian Ministry of Education, University and Research to the “Rita Levi Montalcini” Department of Neuroscience, University of Torino, Italy. Funding sources had no role in design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.