Identification of a FUS splicing mutation in a large family with amyotrophic lateral sclerosis.
ABSTRACT Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease characterized by the degeneration of upper and lower motor neurons. Genetic studies have led, thus far, to the identification of 12 loci and 9 genes for familial ALS (FALS). Although the distribution and impact of superoxide dismutase 1 mutations has been extensively examined for over a decade, the recently identified FALS-associated FUS gene has been less studied. Therefore, we set out to screen our collection of FALS cases for FUS mutations. All 15 exons of FUS were amplified and sequenced in 154 unrelated FALS cases and 475 ethnically matched healthy individuals. One substitution located in the acceptor splice site of intron 14 was identified in all affected members of a large family, causing the skipping of the last 13 amino acids of the protein and the translation of 7 novel amino acids, resulting from the new translation of a part of the 3' untranslated region. Our study identified a new splicing mutation in the highly conserved C-terminal of the FUS protein. Thus far most FUS mutations are missenses, and our findings, combined with those of others, confirm the importance of the C-terminal portion of the protein, adding additional support for FUS mutations having a critical role in ALS.
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ABSTRACT: Mutations in the gene encoding Fused in Sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. FUS is a predominantly nuclear DNA and RNA binding protein that is involved in RNA processing. Large FUS immuno-reactive inclusions fill the perikaryon of surviving motor neurons of ALS patients carrying mutations at post-mortem. This sequestration of FUS is predicted to disrupt RNA processing and initiate neurodegeneration. Here we demonstrate that C-terminal ALS mutations disrupt the nuclear localising signal (NLS) of FUS resulting in cytoplasmic accumulation in transfected cells and patient fibroblasts. FUS mislocalisation is rescued by the addition of the wild-type FUS NLS to mutant proteins. We also show that oxidative stress recruits mutant FUS to cytoplasmic stress granules where it is able to bind and sequester wild-type FUS. While FUS interacts with itself directly by protein-protein interaction, the recruitment of FUS to stress granules and interaction with PABP is RNA dependent. These findings support a two-hit hypothesis whereby cytoplasmic mislocalisation of FUS protein, followed by cellular stress contributes to the formation of cytoplasmic aggregates that may sequester FUS, disrupt RNA processing and initiate motor neuron degeneration.Human Molecular Genetics 03/2013; · 7.69 Impact Factor
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ABSTRACT: Systematic review data demonstrate that 5% of ALS cases are familial (FALS). Causative superoxide dismutase-1 (SOD1) mutations are identified in 10-20% of FALS. Few reports of FALS epidemiology exist in Canada. We completed a retrospective review of all FALS cases within the province of Alberta between 2002 and 2011. Descriptive summaries of genotypes identified and calculation of prevalences were performed. We reviewed 946 clinic database records and 49 subjects with FALS were identified (5.2%). Clinic charts for 47/49 were available and reviewed. Causative SOD1 mutations were observed in 17/47 (36%). The SOD1 I113T mutation was identified in 11/47 unrelated patients and was associated with a less variable survival than previously reported. The period and point prevalences of FALS in Alberta are approximately 2.05 per 100,000 (95% CI 1.51-2.73) and 4.68 per 1,000,000 (95% CI 2.42-8.18), respectively. In conclusion, we report 47 cases of FALS in Alberta over the past decade. The proportion of SOD1-positive FALS cases is higher than reported elsewhere. The high proportion of I113T mutations is comparable to that previously observed in the adjacent province of British Columbia.Amyotrophic lateral sclerosis and frontotemporal degeneration. 05/2013; 14(4):273-277.
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ABSTRACT: Mutations in the TAR DNA Binding Protein gene (TARDBP), encoding the protein TDP-43, were identified in amyotrophic lateral sclerosis (ALS) patients. Interestingly, TDP-43 positive inclusion bodies were first discovered in ubiquitin-positive, tau negative ALS and frontotemporal dementia (FTD) inclusion bodies, and subsequently observed in the majority of neurodegenerative disorders. To date, 47 missense and one truncating mutations have been described in a large number of familial (FALS) and sporadic (SALS) patients. Fused in Sarcoma (FUS) was found to be responsible for a previously identified ALS6 locus, being mutated in both FALS and SALS patients. TARDBP and FUS have a structural and functional similarity and most of mutations in both genes are also clustered in the C-terminus of the proteins. The molecular mechanisms through which mutant TDP-43 and FUS may cause motor neuron degeneration are not well understood. Both proteins play an important role in mRNA transport, axonal maintenance and motor neuron development. Functional characterization of these mutations in in vitro and in vivo systems is helping to better understand how motor neuron degeneration occurs. This report summarizes the biological and clinical relevance of TARDBP and FUS mutations in ALS. All the data reviewed here has been submitted to a database based on the Leiden Open (source) Variation Database(LOVD) and is accessible online at www.lovd.nl/TARDBP, www.lovd.nl/FUS.Human Mutation 04/2013; · 5.21 Impact Factor
Identification of a FUS splicing mutation in a large
family with amyotrophic lateral sclerosis
Ve ´ronique V Belzil1, Judith St-Onge1, Hussein Daoud1, Anne Desjarlais1, Jean-Pierre Bouchard2,
Nicolas Dupre ´2, William Camu3, Patrick A Dion1,4and Guy A Rouleau1,5,6
Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease characterized by the degeneration of upper and lower
motor neurons. Genetic studies have led, thus far, to the identification of 12 loci and 9 genes for familial ALS (FALS). Although
the distribution and impact of superoxide dismutase 1 mutations has been extensively examined for over a decade, the recently
identified FALS-associated FUS gene has been less studied. Therefore, we set out to screen our collection of FALS cases for
FUS mutations. All 15 exons of FUS were amplified and sequenced in 154 unrelated FALS cases and 475 ethnically matched
healthy individuals. One substitution located in the acceptor splice site of intron 14 was identified in all affected members of a
large family, causing the skipping of the last 13 amino acids of the protein and the translation of 7 novel amino acids, resulting
from the new translation of a part of the 3¢ untranslated region. Our study identified a new splicing mutation in the highly
conserved C-terminal of the FUS protein. Thus far most FUS mutations are missenses, and our findings, combined with those
of others, confirm the importance of the C-terminal portion of the protein, adding additional support for FUS mutations having a
critical role in ALS.
Journal of Human Genetics (2011) 56, 247–249; doi:10.1038/jhg.2010.162; published online 16 December 2010
Keywords: amyotrophic lateral sclerosis; FUS/motor neuron disease; neurodegeneration; RNA-binding protein; splicing mutation
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease
affecting the upper and lower motor neurons. Specifically, neurons of
the motor cortex, brain stem and spinal cord are progressively
involved, causing gradual spasticity and muscle weakness starting
in the limbs in 75% of cases and in the bulbar region in 25% of
cases. In all, 90% of patients are believed to be sporadic (SALS),
without any family history of the disease, while the other 10% of cases
are familial (FALS), primarily segregating in an autosomal dominant
manner.1SALS and FALS patients are clinically indistinguishable,
except for the mean age of onset, which is 56 years for SALS,
compared with an average of 46 years for FALS.2The overall
prevalence is 4–6/100000 and incidence is 1–2/100000,3which
make ALS the most common of motor neuron diseases.
Mutations in the copper superoxide dismutase 1 gene were
first shown to be ALS causative over 15 years ago and they account
for 15–20% of all FALS cases, representing a proportion of 1–2% of all
ALS cases.4In the last 2 years, the identification of ALS causative
mutations in the TAR-DNA-binding protein (TARDBP encoding
TDP-43) gene in both SALS and FALS cases5and in FUS/TLS
encoding FUS,6,7thus far mostly but not exclusively in FALS, opened
a new era for the investigation of mechanisms underlying the disease.
Whereas superoxide dismutase 1 mutations have been reported
throughout the full length of the protein, TARDBPand FUS mutations
are mostly clustered to specific regions. For TARDBP, most of the
mutations identified are in the glycine-rich region encoded by exon 6,
and for FUS/TLS, which also contains a glycine-rich region, mutations
are mostly in the extreme C-terminal part of the protein.8The aim of
this study was to evaluate the proportion of FUS mutations in a
portion of our FALS cohort and to see if any mutations identified
would be clustered to the same region.
The 15 coding exons of FUS were amplified and sequenced in 154
unrelated FALS cases, and only one variant was identified. It is an
unreported substitution of an adenine to a cytosine in the acceptor
splice site of intron 14 (c.1542-2A4C). The variant was present in six
samples of a large ALS family (Figure 1) recruited in France: II:5, III:8,
IV:1, IV:3, IV:4, and IV:6. Two of those individuals had an ALS
phenotype (II:5, III:8), while the four unaffected mutation carriers
are in the fourth generation and are younger than the average age of
onset of the first ALS symptoms. By carrying the mutation, they
confirm that their three affected fathers likely had the substitution.
In addition, three mothers married to affected fathers (II:4, III:1 and
III:7) were tested and were negative for the mutation. We prepared
cDNA using total RNA from the immortalized lymphoblast cell
lines of the two affected individuals with the adenine-to-cytosine
Received 30 October 2010; revised 18 November 2010; accepted 26 November 2010; published online 16 December 2010
1Center of Excellence in Neuromics of Universite ´ de Montre ´al, CHUM Research Center, Montreal, Quebec, Canada;2Universite ´ Laval, Faculty of Medicine, CHA, Enfant-Je ´sus
Hospital, Quebec, Canada;3ALS Center, Department of Neurology, CHU Gui de Chauliac, Montpellier, France;4Department of Pathology and Cellular Biology, Faculty of Medicine,
Universite ´ de Montre ´al, Montreal, Quebec, Canada;5Department of Medicine, Faculty of Medicine, Universite ´ de Montreal, Montreal, Quebec, Canada and6Research Center,
CHU Sainte-Justine, and Department of Pediatrics and Biochemistry, Universite ´ de Montreal, Montreal, Quebec, Canada
Correspondence: Dr GA Rouleau, CHUM Research Centre, Notre-Dame Hospital, 1560 Sherbrooke East, Y-3633, Montreal, Quebec, Canada H2L 4M1.
Journal of Human Genetics (2011) 56, 247–249
& 2011 The Japan Society of Human Genetics All rights reserved 1434-5161/11 $32.00
substitution. These cDNAs were PCR amplified, and two products of
425 and 167bp were observed on agarose gel (Figure 2a), demonstrat-
ing that the mutant allele was not degraded by nonsense-mediated
mRNA decay. The heterozygous sequence trace was analyzed, as
well as the separate sequence of the two different alleles after a gel
extraction of the two products (Figure 2b). Sequencing showed that
patients with the c.1542-2A4C change expressed a mutated allele
missing the 40bp coding sequence of exon 15 as well as the first 203bp
of the 3¢ untranslated region. More precisely, the sequence of
the mutant mRNA brings together the last 2bp of amino acid 514
located in exon 14 and the nucleotide at the c.*204 position in the
3¢ untranslated region, as an AG alternative acceptor site located at
position c.*202_*203 is encountered (Figure 2c); translation subse-
quently ends with a stop codon at position c.*228. Consequently, the
mutant protein undergoes a silent change at amino acid 514
(p.R514R), followed by seven new amino acids (SMSRSGR) at the
end of the protein. The adenine of the wild-type acceptor site of intron
14 located at base pair 31110219 on chromosome 16 is highly
conserved across species and is not listed as a known SNP. The
Alternative Splice Site Predictor bioinformatics program attributed a
score of 15.533 to the wild-type AG acceptor splice site, and a score
of 4.249 for the acceptor splice site with the cytosine substitution.
The alternative AG acceptor splice site located in the 3¢ untranslated
region at position c.*202_*203 had a score of 4.620 and is suggested to
Figure 1 Pedigree of the family with the c.1542-2A4C variant. Black
symbols represent affected individuals, while open symbols represent
unaffected family members. Symbols with dots represent unaffected
individuals carrying the substitution. Slashed symbols indicate deceased
individuals. A red square around the individual III:8 symbol indicates the
sample sequenced in the primary screening, and asterisks indicate family
members for whom exon 15 of the FUS gene was sequenced in the
secondary screening. Symbols with two asterisks represent individuals
for whom cDNA was amplified. Genotypes at position c.1542-2 are
indicated in black for the wild-type allele and in red for the mutant allele.
Genotypes in parentheses are inferred.
Figure 2 Splicing mutation in FUS. (a) Agarose gel electrophoresis of FUS cDNA amplified in one unrelated control, and two affected family members
(II:5, III:8), showing the wild-type allele (425bp) and the mutant allele (167bp) for the fragment amplified using foward primer 5¢-GAGGGGG
ACCAGGTGGCTCTCAC in exon 14 and reverse primer 5¢-TCATTTGGCCTTCTCCCCGAACAC in the 3¢ untranslated region (UTR). (b) cDNA sequence
chromatogram of the wild-type and mutant alleles in one patient, confirming the skipping of exon 15 and the first 203bp of the 3¢UTR. (c) Schematic
(not to scale) representation of exons 14, 15 and the 3¢UTR of the FUS gene for the wild-type and mutant alleles. In the mutant allele, a part of the 3¢UTR
is translated, which adds seven new amino acids to the protein (SMSRSGR).
FUS splicing mutation in an ALS family
VV Belzil et al
Journal of Human Genetics
be responsible for this new isoform. As this FUS mutation segregates
in all affected individuals of the family as well as in the children of
deceased affected members, and was not present in 475 control
participants, we believe that it is likely responsible for the ALS
phenotype observed in this family.
The substitution is located in the RGG domain enriched in
arginine–glycine–glycine motifs, where most other FUS mutations
were reported. Importantly, truncated proteins were previously
observed with the two other major ALS causative genes.9–11But,
more interestingly, this is the second splicing mutation reported
in FUS, as another one was recently shown to lead to the skipping
of exon 14 after the substitution of the adenine located in the acceptor
splice site of intron 13.12Unfortunately, no clinical information could
be retrieved for the family presented in our study, so we were unable
to compare the disease evolution caused by our splicing mutation in
intron 14 with the splicing mutation in intron 13 reported by Dejesus-
Hernandez et al.12The splicing mutation identified here removes
the last 13 amino acids that partly encode the C-terminal portion
of FUS, in which a number of mutations have been associated with
ALS. Our finding reinforces the notion that this portion has a key role
in the pathology of ALS, most likely by way of affecting the RNA-
binding domain it encodes for. In the future, such splicing defect
causative mutations may become treatable through drugs or gene
therapies aimed at correcting splicing, as explored for a number
VVB, HD and GAR are supported by the Canadian Institutes of Health Research.
We would like to thank the patients involved in this study; Me ´lanie Benard,
Isabelle Thibault and Pierre Provencher for sample collection and organization;
Anne Noreau, Cynthia Bourassa, Sophie Massart and Bertrand Boutie ´ for
technical support; and to acknowledge support from the Association pour la
Recherche sur la Scle ´rose Late ´rale Amyotrophique (ARS), the Association
Franc ¸aise contre les Myopathies (AFM) and the French Group on MND.
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assay toidentify small molecule
FUS splicing mutation in an ALS family
VV Belzil et al
Journal of Human Genetics