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Mutations in the gene encoding fused in sarcoma (FUS) were recently identified as a novel cause of amyotrophic lateral sclerosis (ALS), emphasizing the genetic heterogeneity of ALS. We sequenced the genes encoding superoxide dismutase (SOD1), TAR DNA-binding protein 43 (TARDBP) and FUS in 99 sporadic and 17 familial ALS patients ascertained at Mayo Clinic. We identified two novel mutations in FUS in two out of 99 (2.0%) sporadic ALS patients and established the de novo occurrence of one FUS mutation. In familial patients, we identified three (17.6%) SOD1 mutations, while FUS and TARDBP mutations were excluded. The de novo FUS mutation (g.10747A>G; IVS13-2A>G) affects the splice-acceptor site of FUS intron 13 and was shown to induce skipping of FUS exon 14 leading to the C-terminal truncation of FUS (p.G466VfsX14). Subcellular localization studies showed a dramatic increase in the cytoplasmic localization of FUS and a reduction of normal nuclear expression in cells transfected with truncated compared to wild-type FUS. We further identified a novel in-frame insertion/deletion mutation in FUS exon 12 (p.S402 P411delinsGGGG) which is predicted to expand a conserved poly-glycine motif. Our findings extend the mutation spectrum in FUS leading to ALS and describe the first de novo mutation in FUS.
Major advances have recently been made in our understanding of the genetic causes underlying amyotrophic lateral sclerosis (ALS) (Dion, et al., 2009; Van Damme and Robberecht, 2009). ALS is a neurodegenerative disorder characterized by the loss of motor neurons in the spinal cord, brain stem, and motor cortex, resulting in progressive muscle weakness and atrophy which is typically fatal within 3–5 years (Boillee, et al., 2006). Most patients with ALS are sporadic, however, a positive family history of ALS has been found in 5–10% of all reported cases (Mitchell and Borasio, 2007). ALS is a genetically complex disorder with multiple causal genes and genetic risk factors contributing to the disease. Mutations in the copper/zinc superoxide dismutase 1 gene (SOD1; MIM# 147450) are the most common cause of ALS and account for about 15–20% of familial adult-onset patients with ALS and 1–2% of sporadic patients (Dion, et al., 2009; Rosen, et al., 1993). More recently, mutations in the genes encoding the TAR DNA-binding protein 43 (TARDBP; MIM# 605078) and fused in sarcoma/translocated in liposarcoma (FUS/TLS; MIM# 137070), two multifunctional DNA/RNA binding proteins, were identified in familial ALS with subsequent reports of mutations in sporadic patients (Kabashi, et al., 2008; Kwiatkowski, et al., 2009; Lagier-Tourenne and Cleveland, 2009; Sreedharan, et al., 2008; Vance, et al., 2009). Several other less common gene mutations linked to familial adult-onset ALS with or without frontotemporal dementia (FTD; MIM# 600274) have been reviewed elsewhere (Dion, et al., 2009). An important locus of the combined phenotype of ALS and FTD has been mapped to a region on chromosome 9p (Le Ber, et al., 2009; Morita, et al., 2006; Vance, et al., 2006); however, the causative gene has not yet been identified.
FUS/TLS is a member of the TET family of RNA binding proteins, which also includes Ewing’s sarcoma (EWSR1; MIM# 133450) and TATA-binding associated factor (TAF15; MIM# 601574) (Tan and Manley, 2009). The FUS gene is located on chromosome 16p11.2 and consists of 15 exons encoding a protein of 526 amino acid residues (Morohoshi, et al., 1998). Like other TET proteins, FUS has an N-terminal transcriptional activation domain enriched in glutamine, glycine, serine, and tyrosine (QGSY) residues, a glycine-rich region and several structural motifs involved in RNA-binding, including an RNA-recognition motif (RRM), multiple arginine-glycine-glycine (RGG) repeats and a putative zinc finger domain, at the C-terminus (Iko, et al., 2004). FUS binds RNA in vitro and in vivo (Crozat, et al., 1993; Zinszner, et al., 1997) and is involved in several mRNA-processing steps, such as transcription regulation (Uranishi, et al., 2001), RNA splicing (Meissner, et al., 2003; Yang, et al., 1998), and RNA transport including nucleo-cytoplasmic shuttling (Zinszner, et al., 1997). FUS also binds DNA and plays a role in DNA repair through homologous DNA pairing and recombination (Baechtold, et al., 1999) and acts as a transcriptional regulatory sensor of DNA damage signals to assure genomic integrity (Wang, et al., 2008). FUS deficient mice indeed show high levels of chromosomal instability and perinatal mortality (Hicks, et al., 2000) and an increased sensitivity to ionizing radiation (Kuroda, et al., 2000). Through chromosomal translocations, the N-terminus of FUS functions as an essential transforming domain for a number of fusion oncoproteins implicated in the development of human liposarcomas and leukemias (Law, et al., 2006). FUS shows an almost ubiquitous nuclear expression but is also present in the cytoplasm of most cell types (Andersson, et al., 2008). In neurons, FUS is predominantly localized to the nucleus (Andersson, et al., 2008). Interestingly, neuropathological analysis of brain and spinal cord of ALS patients carrying FUS mutations showed cytoplasmic retention and the formation of FUS and ubiquitin-positive neuronal aggregates, although at least some preservation of normal physiological nuclear FUS immunoreactivity was observed (Kwiatkowski, et al., 2009; Vance, et al., 2009).
In less than one year, 23 different mutations have been identified in FUS in 38 families and 12 sporadic ALS patients (Belzil, et al., 2009; Blair, et al., 2009; Chio, et al., 2009; Corrado, et al., 2009; Drepper, et al., 2009; Kwiatkowski, et al., 2009; Rademakers, et al., in press; Ticozzi, et al., 2009; Van Damme, et al., 2009; Vance, et al., 2009). The majority of the mutations are missense changes affecting the highly conserved most C-terminal part of FUS; however missense changes and one in-frame deletion have also been reported in the QGSY and glycine-rich region. The reported mutation frequencies of FUS in familial ALS range from 1–5%, with lower estimates (0–1%) in populations of sporadic ALS patients (Belzil, et al., 2009; Blair, et al., 2009; Chio, et al., 2009; Corrado, et al., 2009; Drepper, et al., 2009; Kwiatkowski, et al., 2009; Rademakers, et al., in press; Ticozzi, et al., 2009; Van Damme, et al., 2009; Vance, et al., 2009). Here we aimed to determine the spectrum and contribution of SOD1, TARDBP and FUS mutations in a newly ascertained Mayo Clinic ALS series and identified three known SOD1 mutations in familial ALS patients (17.6%) and two novel FUS mutations in sporadic ALS patients (2.0%), including one patient with a de novo FUS mutation. Importantly, we identified a splice-site mutation defining a new category of FUS mutations characterized by C-terminal truncation of FUS. Using cellular expression studies we show a remarkable increase in cytoplasmic localization of truncated FUS compared to full-length wild-type FUS, supporting the presence of a nuclear localization signal (NLS) within the C-terminus of FUS.
Our study cohort consisted of 108 unrelated ALS patients and 9 patients with other diagnosis (61 males, 56 females) from a consecutive clinical case series seen at Mayo Clinic Florida by the ALS Center in the period 2008–2009. All patients agreed to be in the study and biological samples were obtained after informed consent. Each patient underwent a full neurological evaluation including electromyography, clinical laboratory testing and imaging as appropriate to establish the clinical diagnosis of ALS. A total of 108 patients were diagnosed with ALS according to El Escorial criteria, 6 patients were diagnosed with primary lateral sclerosis (PLS), 1 patient with progressive muscular atrophy (PMA) and 2 patients with monomelic amyotrophy. In the ALS patient cohort, 15.7% of patients (17/108) showed a positive family history of ALS defined as having at least one affected relative within 3 generations. The disease onset was bulbar in 23.1% of ALS patients and spinal in 76.9%. Thirty-seven patients (34.3%) showed frontotemporal cognitive impairment and/or pseudobulbar affect. The average age of onset in our complete patient population was 56.6 ± 10.9 years (range 20–78 years). DNA samples of 700 healthy control individuals (range 30–99 years) were also ascertained at the Department of Neurology at Mayo Clinic Florida.
Genomic DNA (gDNA) was extracted from blood samples using standard automation protocols with the AutoGenprep 245T (Autogen, Holliston, MA, USA). For each patient, polymerase chain reaction (PCR) amplification was performed for all 15 exons of FUS, all 5 exons of SOD1 and exon 6 of TARDBP, using primers designed to flanking intronic sequences using Qiagen products (Qiagen, Valencia, CA, USA) (PCR conditions and primer sequences available on request). PCR products were purified using the Ampure system (Agencourt Bioscience Corporation, Beverly, MA, USA) and sequenced using Big dye terminator V.3.1 products (Applied Biosystems, Foster City, CA, USA). Sequencing products were purified using the CleanSEQ method (Agencourt) and analyzed on an ABI 3730 DNA analyzer (Applied Biosystems). Sequence analysis was performed using Sequencher software (Gene Codes, Ann Arbor, MI, USA). We determined the presence of the g.10747A>G mutation in control individuals by PCR and sequencing analysis of exon 14 of FUS. The presence of the complex g.10096_10124delinsGGAGGTGGAGG mutation in exon 12 of FUS in control individuals was performed by PCR amplification of exon 12 of FUS using one fluorescently labeled primer (Ex12F-FAM-GGAGCAGACCCATACTTG and Ex12R-ACACGCACATACCTCAGTAC) followed by fragment length analysis on an automated ABI3730 DNA-analyzer (Applied Biosystems). The 18-bp difference between the wild-type and mutant allele was scored using GENEMAPPER v4.0 (Applied Biosystems). Haplotype sharing analysis of six short tandem repeat (STR) markers on chromosome 16 flanking FUS (D16S401, D16S3068, D16S769, D16S753, D16S411 and D16S3136) in family ALS046 was also analyzed by GENEMAPPER.
RNA extraction for patient ALS046-01, 8 control individuals and 7 unrelated ALS patients without FUS mutations was performed from 2-ml frozen blood aliquots collected in EDTA tubes, using a modified version of the protocol described by Beekman et al. (Beekman, et al., 2009). In short, the blood was thawed on ice for 2–3h and transferred to a PAXgene RNA collection tube (PreAnalytix; Qiagen, Valencia, CA, USA). After an incubation of 16–21h at room temperature we extracted RNA using the PAXgene RNA extraction kit (PreAnalytix) using the protocol recommended by the manufacturer. RNA concentrations were measured using a NanoDrop (Thermoscientific, Wilmington, DE, USA) and ranged from 10 to 100 ng/µl. First-strand synthesis was carried out using the Superscript III system (Invitrogen, Carlsbad, CA, USA), with 70–400 ng of total RNA as the template and a combination of random hexamers and oligo(dT) primers. The resultant complementary DNA (cDNA) was used as the template for RT-PCR reactions (Qiagen) using the following primer pairs: cDNA12/13F-GTCCTAATCCCACCTGTGAG and utrR-CTTGGGTGATCAGGAATTG. RT-PCR reactions were denatured for 3 minutes at 94 °C, then cycled at 60-50 °C touchdown (30”, 30”, 45”) for 35 cycles. RT-PCR products were visualized by electrophoresis on 1.5 % agarose gels, gel-excised, then DNA extracted using the QIAquick Gel Extraction Kit (Qiagen) and sequenced in both directions as described above.
A human cDNA clone encoding full-length wild type FUS (Invitrogen clone #2822692) was used as a PCR template to generate V5-tagged wild-type FUS (V5-FUSWT) and p.R521C mutant FUS (V5-FUSR521C) cDNA using primers listed in Supp. Table S1. cDNA containing p.G466VfsX14 truncated FUS, predicted to result from the deletion of FUS exon 14 (V5-FUSΔ14), was generated by PCR using cDNA of patient ALS046-01 as a template and the same primer set used to generate V5-FUSWT. Inserts were cloned into the pAG3 mammalian expression vector using XhoI and BamHI restriction sites. The sequences of all pAG3 FUS expression constructs were confirmed by direct sequencing of the complete cDNA inserts and flanking vector sequences.
N2A (mouse neuroblastoma cell line) cells were cultured in Dulbecco's Modified eagles medium (DMEM) supplemented with 10% fetal bovine serum. For immunofluorescence microscopy, N2A cells were plated on 13mm diameter glass coverslips in a 24-well plate and transfected using 0.2% Lipofectamine2000 (Invitrogen) with 600ng V5-FUSWT, V5-FUSR521C, or V5-FUSΔ14 respectively. After 36 hours, cells were fixed with 4% paraformaldehyde for immunostaining. The fixed cells were blocked for 1 hour with 5% BSA and subsequently incubated with 1:500 of primary antibody (mouse anti-V5 antibody, Invitrogen) for 1 hour. Fluorescent signal was detected using 1:1000 dilution of Alexa Fluor 488-labeled donkey anti-mouse antibody (Invitrogen). The cells were observed using 100× magnification by confocal microscopy. For Western blot analysis, N2A cells were plated in 6-well dishes and transfected using 0.2% Lipofectamine2000 (Invitrogen) with 2µg V5-FUSWT, V5-FUSR521C, or V5-FUSΔ14 respectively. Nuclear and cytoplasmic enriched protein fractions were isolated from the cells 48 hours post-transfection using the ProteoJET™ Cytoplasmic and Nuclear Protein Extraction Kit (Fermentas, Ontario, Canada) following the manufacturer’s protocol. Total protein concentrations of the cytosolic and nuclear fractions were quantified using the bicinchoninic acid (BCA) assay (Pierce). 30µg of protein from the nuclear fraction and 8µg of protein from the cytosolic fractions were separated on 10% SDS-PAGE gels, transferred to PVDF membranes and blocked for 1 h at RT with 5% skim milk/TBST. V5-FUS proteins were detected with anti-V5 primary antibody (1:5000; Invitrogen) followed by anti-mouse HRP conjugated secondary antibody (1:5000; Jackson Immuno Research, West Grove, PA) and ECL-Plus (Perkin Elmer, Waltham, MA). Re-probing of the blots with anti-GAPDH (1:5000; Biodesign International, Saco, ME) and anti-H3 (1:1000; Cell Signaling, Danvers, MA) was performed to confirm enrichment and equal loading of the cytosolic and nuclear fractions.
We performed systematic mutation analyses of SOD1, TARDBP and FUS through direct sequencing in 117 patients diagnosed with ALS, PLS, PMA or monomelic amyotrophy from a consecutive clinical case series ascertained at Mayo Clinic Florida. Our sequence analysis identified two novel mutations in FUS in two sporadic ALS patients (Figure 1) and three known mutations in SOD1 in three familial ALS patients (Table 1). TARDBP mutations were not identified in our patient series. The population frequency of SOD1 mutations was thus 17.6% of familial ALS (3/17), while FUS mutations were absent from familial patients but accounted for 2.0% of sporadic ALS (2/99). A summary of synonymous coding changes and genetic variants identified in the 3’untranslated regions of SOD1 and FUS is provided in Supp. Table S2.
A heterozygous splice-site mutation, g.10747A>G (IVS13-2A>G), at the splice acceptor site of FUS intron 13 was identified in one sporadic ALS patient (ALS046-01) (Figure 2A). This mutation was predicted to affect the correct splicing of FUS exon 14. Frozen blood of this patient was available as a source of mRNA to study the effect of this mutation on FUS splicing. Amplification of FUS cDNA showed evidence of an aberrant product corresponding to the skipping of exon 14 (148bp) in addition to the full-length wild-type FUS transcript, which was further confirmed by gel-excision and sequencing analysis (Figure 2B). No aberrant PCR products were identified by cDNA analysis of 8 control individuals or 7 ALS patients without FUS mutations. The exclusion of exon 14 (c.1394_1541del) in patient ALS046-01 is expected to result in a frameshift and a premature termination codon (PTC) in exon 15 leading to a truncated FUS protein (p.G466VfsX14) lacking most of the RGG-domain enriched in arginine-glycine-glycine motifs. A complex heterozygous 29-bp deletion and 11-bp insertion mutation in FUS exon 12, g.10096_10124delinsGGAGGTGGAGG (c.1204_1232delinsGGAGGTGGAGG), was identified in another sporadic ALS patient (ALS058-01) (Figure 2C). This mutation is predicted to lead to the deletion of ten amino acids and the insertion of four novel glycine residues within an evolutionarily conserved glycine-rich region (p.S402_P411delinsGGGG) (Figure 2D). Both novel FUS mutations were excluded from 700 healthy control individuals.
Patient ALS046-01 w as a 20-year-old woman who developed progressive dysarthria and dysphagia accompanied by tongue atrophy and fasciculations. Cervical, upper and lower limb weakness bilaterally followed, with diffuse muscle atrophy and widespread fasciculations leading to mutism, dysphagia, head drop and severe difficulty handling oral secretions. There were no definite upper motor neuron signs. Clinical findings supported a diagnosis of El Escorial clinically suspected ALS. A feeding tube was placed 11 months after onset; noninvasive ventilation (NIV) began at 14 months. Limited airway protection led to tracheostomy at 15 months. She died of ventilatory failure 22 months after onset of weakness. Autopsy was not performed.
Patient ALS058-01 was a 65-year-old man who developed exertional dyspnea with atrophy and fasciculations in tongue, trunk, and limb muscles bilaterally. Bulbar poliomyelitis at age 6 years had left him with mild dysphagia but no other motor symptoms. He was recently retired from a job requiring routine physical labor. Increasing respiratory muscle weakness and dysphagia led to initiation of NIV 9 months after onset of weakness, and a feeding tube at 11 months. Clinical findings supported a diagnosis of El Escorial clinically probable laboratory supported ALS. Weakness progressed to ventilatory failure and death 13 months after onset of weakness. Autopsy was not performed. Weakness was more pronounced proximally in both patients. Neither patient exhibited overt cognitive impairment or pseudobulbar affect. In both patients, EMG findings were compatible with ALS, and brain and spinal MRI and laboratory evaluation identified no alternative cause.
Both FUS mutation carriers identified in this study presented without any known family history of motor neuron disease or dementia. The parents of ALS046-01 are currently 53 years of age and healthy. To determine whether ALS046-01 inherited the g.10747A>G mutation from one of her unaffected parents, or whether the mutation occurred de novo, we obtained DNA samples from her father (ALS046-02) and mother (ALS046-03) and performed sequencing analysis of FUS exon 14 and flanking intronic sequences. These analyses showed that the g.10747A>G mutation was not present in the DNA of either one of the parents (Figure 2E). Haplotype analysis using six short tandem repeat markers flanking FUS further supported their genetic kinship, suggesting that the g.10747A>G mutation occurred de novo in ALS046-01. Since both parents of patient ALS058-01 are deceased, we could not evaluate the origin of the FUS mutation in this patient.
To assess the functional importance of the truncated FUS transcript identified in this study, we created mammalian expression vectors expressing N-terminal V5-tagged wild-type FUS (V5-FUSWT), the previously reported p.R521C mutant FUS (V5-FUSR521C), and p.G466VfsX14 truncated FUS, predicted to result from the deletion of FUS exon 14 (FUSΔ14). Transient transfection of V5-FUSWT, V5-FUSR521C, and V5-FUSΔ14 in N2A cells showed a predominant nuclear localization of V5-FUSWT in all transfected cells, while mutant V5-FUSR521C and V5-FUSΔ14 generally showed dense cytoplasmic fluorescent signal with a correspondingly dramatic loss of detection in the nucleus (Figure 3A). In line with previous reports, a small fraction of cells transfected with V5-FUSR521C retained the predominant nuclear localization of FUS. In contrast, predominant nuclear localization of V5-FUSΔ14 was rarely observed. The dramatic increase in cytoplasmic localization of V5-FUSR521C and particularly V5-FUSΔ14 compared to V5-FUSWT was further confirmed by immunoblot analysis of cytosolic and nuclear enriched fractions obtained by subcellular fractionation of N2A cells transfected with each construct (Figure 3B). This analysis also showed the predicted smaller molecular weight band for V5-FUSΔ14 compared to V5-FUSWT and V5-FUSR521C, consistent with the C-terminal truncation of FUS.
We report the identification of two novel FUS mutations as the cause of ALS in two sporadic patients from the Mayo Clinic Florida ALS series. Our findings broaden the mutational spectrum of FUS in ALS to include a complex in-frame insertion/deletion mutation affecting multiple amino acid residues (p.S402_P411delinsGGGG) and a splice-site mutation leading to the C-terminal truncation of FUS (p.G466VfsX14). Mutation and segregation analysis performed in relatives of one mutation carrier further revealed the de novo occurrence of the FUS mutation, explaining the sporadic nature of the disease in this family.
Mutations in the gene encoding the multifunctional DNA/RNA-binding protein FUS were identified earlier this year as the cause of familial ALS type 6 and have been suggested to account for 1–5% of familial ALS (Belzil, et al., 2009; Blair, et al., 2009; Chio, et al., 2009; Corrado, et al., 2009; Drepper, et al., 2009; Kwiatkowski, et al., 2009; Rademakers, et al., in press; Ticozzi, et al., 2009; Van Damme, et al., 2009; Vance, et al., 2009). FUS mutations have also been reported in a total of 12 sporadic patients although several studies failed to identify FUS mutations in sporadic patients (Blair, et al., 2009; Kwiatkowski, et al., 2009) and mutation frequencies were <1% in all sporadic ALS populations studied (Belzil, et al., 2009; Corrado, et al., 2009; Drepper, et al., 2009). These mutation frequencies however may have been underestimated since analysis was restricted in all (Drepper, et al., 2009) or in a subset (Belzil, et al., 2009; Corrado, et al., 2009; Rademakers, et al., in press) of patients to exons previously found mutated in ALS. In our Mayo Clinic ALS series we performed mutation analysis of all FUS exons and identified mutations at a slightly higher frequency, 2 out of 99 sporadic patients or 2.0% of our population. Both mutations were located outside of exon 15, including one mutation in exon 12, an exon not previously implicated in ALS. In contrast, in 17 familial ALS patients we identified three SOD1 mutation carriers (17.6%) but no mutations in FUS; however, these results may be biased due to the small sample size of this population. Despite published TARDBP mutation frequencies of ~3% in familial ALS and ~1.5% in sporadic ALS, we did not identify mutations in exon 6 of TARDBP in any of our ALS patients.
In contrast to the previously published FUS missense mutations and a single codon in-frame deletion (Figure 1), the present study describes a new type of FUS mutations characterized by the C-terminal truncation of FUS. Mutation g.10747A>G (IVS13-2A>G) identified in patient ALS046-01 results in skipping of exon 14 from the FUS transcript leading to a premature termination codon in exon 15 eliminating the C-terminus of FUS including most of the RGG-domain (p.G466VfsX14). Using transcript analysis we confirmed the effect of this mutation on mRNA splicing and determined that this transcript was not subject to nonsense-mediated decay. Our findings emphasize the importance of the in-vivo validation of the effect of mutations on mRNA splicing, especially for newly identified disease genes. It is conceivable that some of the previously published FUS mutations, including g.10895A>G (p.R514G), g.11185G>C (p.R514S) and the double point mutation g.11185G>T; g.11186G>T (p.R514S; p.G515C) may also induce aberrant FUS splicing and the production of truncated FUS proteins. Similarly, the current mutation frequency of FUS in familial and sporadic ALS may be underestimated because out-of frame exonic deletions or duplications resulting in truncated FUS proteins could have been missed in routine gDNA sequencing analyses.
We further identified a complex 29-bp deletion and 11-bp insertion mutation (g.10096_10124delinGGAGGTGGAGG) in exon 12 in patient ALS058-01 predicted to result in the in-frame deletion of ten amino acids and the insertion of four novel glycine residues within a glycine-rich region (p.S402_P411delinsGGGG). Although we were unable to demonstrate segregation of this mutation with disease, we believe this mutation is likely pathogenic since it was not identified in 700 control individuals and it affects evolutionary conserved residues within a functionally critical region of the FUS protein (Figure 2D). In fact, this is the sixth FUS mutation localized to a glycine-rich region identified in sporadic ALS patients, lending support for the recent hypothesis that mutations within glycine-rich regions of FUS may represent low-penetrance mutations (Corrado, et al., 2009).
The clinical presentation of the FUS mutation carriers identified in our series was consistent with classical ALS with a bulbar onset in both patients, except that ALS046-01 did not develop clinical upper motor neuron signs. Absence of upper motor neuron signs has been reported in other patients with FUS mutations but appears to be uncommon (Chio, et al., 2009; Kwiatkowski, et al., 2009; Ticozzi, et al., 2009; Vance, et al., 2009). The phenotype otherwise was similar to that reported in other series (Belzil, et al., 2009; Chio, et al., 2009; Kwiatkowski, et al., 2009; Ticozzi, et al., 2009; Vance, et al., 2009). Interestingly, the onset ages of our two patients differed significantly with an extreme early onset (20 years) in ALS046-01 carrying the FUS truncation mutation, which may be the result of its severe effect on FUS localization and function. However, a wide variability in onset ages has previously been observed in FUS mutation carriers (Belzil, et al., 2009; Blair, et al., 2009; Chio, et al., 2009; Kwiatkowski, et al., 2009; Ticozzi, et al., 2009; Van Damme, et al., 2009; Vance, et al., 2009), even within patients carrying the same mutation, suggesting that additional genetic and/or environmental factors may be important in defining the age-related disease penetrance of FUS mutations.
Both FUS mutation carriers identified in this study presented with sporadic ALS. For one patient, ALS046-01, we were able to obtain DNA samples from her unaffected parents which revealed a de novo occurrence of the FUS mutation in this family. Unfortunately, like all previously reported sporadic FUS mutation carriers, relatives of ALS058-01 were unavailable for study such that we could not distinguish between a de novo mutation and reduced penetrance in this family. Interestingly, a de novo SOD1 mutation was previously reported as the cause of young-onset sporadic ALS (Alexander, et al., 2002). Together these studies suggest de novo mutations may be more important than previously recognized. Demonstration of a de novo FUS mutation in a sporadic ALS patient in this study emphasizes that mutational analysis should be considered in isolated instances of sporadic ALS patients, in addition to those with a positive family history.
A limited characterization of the pathology associated with mutations in FUS was previously reported showing a partial retention of FUS in the cytoplasm and the formation of FUS and ubiquitin-positive neuronal aggregates (Kwiatkowski, et al., 2009; Vance, et al., 2009). Also, in cell culture, mislocalization of mutant FUS was demonstrated for at least two C-terminal missense mutations (p.R521C, p.R521G) (Kwiatkowski, et al., 2009; Vance, et al., 2009). These findings, combined with previous data on the identification and characterization of the nuclear localization signal (NLS) in EWSR1, another TET family member highly homologous to FUS in the C-terminal region, suggests that the 18 most C-terminally located amino acids of FUS comprise a non-conventional NLS required for the nuclear import and retention of FUS (Zakaryan and Gehring, 2006). In this scenario, all previously published FUS missense mutations clustered at the extreme C-terminus of FUS would directly affect the NLS sequence, while the truncation mutation (p.G466VfsX14) identified in this study would completely eliminate the NLS. Immunofluorescence assays in N2A cells transfected with V5-FUSΔ14, V5-FUSR521C, and V5-FUSWT indeed showed a dramatic increase in cytoplasmic FUS localization and a reduction of nuclear detection in cells transfected with V5-FUSR521C and V5-FUSΔ14 compared to V5-FUSWT. Immunoblot analysis of nuclear and cytoplasmic enriched protein fractions further confirmed the cytoplasmic accumulation of mutant FUS, and suggested a more severe mislocalization in cells transfected with V5-FUSΔ14 compared to V5-FUSR521C. Autopsy tissue was not available for the FUS truncation mutation carrier to confirm these effects in vivo. Whether the mislocalization of FUS is critical in the disease pathogenesis, either through the loss of one of the normal nuclear functions of FUS in mRNA processing and transport or from the toxic effect of the cytoplasmic aggregates, remains unclear.
The identification of a truncating FUS mutation and a complex insertion/deletion mutation in a conserved poly-glycine motif in two sporadic ALS patients from our series extends the spectrum of FUS mutations and underlines the importance of extensive population-based mutation screening of newly identified genes. Additional FUS mutation screenings, including cDNA transcript analysis, should lead to a better understanding of the complete spectrum and frequency of FUS mutations and the contribution of de novo mutations in FUS to sporadic ALS. Further in depth in vitro and in vivo studies are needed to elucidate the pathological mechanism underlying ALS and related FUS proteinopathies.
This work was supported by NIH/NIA grant AG26251-03A1 (Rosa Rademakers), the ALS association (Rosa Rademakers), the Pacific Alzheimer Research Foundation (PARF) (Rosa Rademakers, Zbigniew K. Wszolek), Mayo Clinic Florida ALS Center donor funds, and the Robert and Clarice Smith Fellowship program (Jannet Kocerha). Rosa Rademakers is also partially funded by R01 NS065782 and P50 AG16574. Zbigniew K Wszolek is partially funded by P01 AG017216, R01 NS057567, R01 AG015866, and CIHR 121849. We thank Karen Jansen and Christophe Verbeeck for assisting in the cloning and Caroline Kent for technical assistance with the confocal microscopy. This paper is dedicated to the patients and families who participated in this work.