P525L Fused-in-Sarcoma (FUS) mutation is associated with a specific amyotrophic lateral sclerosis (ALS) phenotype characterized by a juvenile-onset and a severe course. 1 This harmful point mutation is located in the nuclear localization signal (NLS) domain at the protein C-terminal. 2 Although wild-type FUS protein is expressed almost exclusively in the nucleus, the P525L FUS mutation leads to a protein mislocalization into the cytoplasm 3,4 because of its loss of capacity to bind its transporter karyopherin-2 and to be transferred back to the nucleus. 3 Here, we compare FUS expression and localization in skin fibroblasts of 2 sisters, both carriers of a P525L FUS mutation, belonging to a Sicilian family with this mutation. 4,5The first sister (DC) was seen at age 21 years when she was asymptomatic. After almost 2-year follow-up, she developed a bulbar form of ALS and died 13 months after disease onset. In both conditions, i.e., asymptomatic (DC-A, at the time of the first visit) and symptomatic (DC-S, soon after disease onset), skin fibroblasts were purified and cultured. The other sister (DL) was seen when she was aged 25 years; on that occasion, fibroblasts were also purified. She is at present asymptomatic.We studied the expression and subcellular localization of FUS protein in fibroblasts from the 2 P525L FUS carriers. Concerning DC, we analyzed FUS expression in the fibroblasts when she was asymptomatic and after the disease onset. A patient with sporadic clinically definite ALS with no known ALS-related gene mutations (sporadic ALS [S-ALS]) and a healthy control (HC) were used as controls.All individuals and patients involved in this study signed informed consent for the genetic testing and the skin biopsy. The experimental protocol was approved by the Ethics Committee Palermo 1 (July 2017).Fibroblasts were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% calf serum and antibiotic/antimycotic solution. Cells were plated on glass coverslips to perform immunofluorescence by using a polyclonal FUS antibody (11570-1-AP, Proteintech Group, Chicago, IL). The analysis of subcellular FUS expression was made through a Zeiss LSM5 confocal microscope. Cell counting for subcellular FUS expression was performed as described by Lo Bello et al. 4 As expected, FUS mislocalized to the cytoplasm in almost all fibroblasts carrying the P525L FUS mutation. 4 Conversely, control fibroblasts (S-ALS and HC) expressed FUS only in the nucleus (figure, A).After a more careful inspection, we observed important differences in the nucleuscytoplasm distribution of FUS protein between the 2 FUS mutants (DC and DL). By visual