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.

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

Canosa A.
First
;
Lomartire A.;De Marco G.;Grassano M.;Brunetti M.;Manera U.;Vasta R.;Salamone P.;Fuda G.;Sbaiz L.;Gallone S.;Moglia C.;Calvo A.;Chio A.
2021

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.
N/A
N/A
Amyotrophic lateral sclerosis; FUS; loss of function; splice site mutation
Canosa A.; Lomartire A.; De Marco G.; Grassano M.; Brunetti M.; Manera U.; Vasta R.; Salamone P.; Fuda G.; Sbaiz L.; Gallone S.; Moglia C.; Calvo A.; Chio A.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/1805324
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