Identification of a FUS splicing mutation in a large family with amyotrophic lateral sclerosis
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.
Identiﬁcation of a FUS splicing mutation in a large
family with amyotrophic lateral sclerosis
´ronique V Belzil1, Judith St-Onge1, Hussein Daoud1, Anne Desjarlais1, Jean-Pierre Bouchard2,
´2, William Camu3, Patrick A Dion1,4 and 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 (TA RDB P encoding
TDP-43) gene in both SALS and FALS cases5and in FUS/TLS
encoding FUS,6,7 thus 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, TAR D B P and FUS mutations
are mostly clustered to speciﬁc regions. For TAR D BP,mostofthe
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
´al, CHUM Research Center, Montreal, Quebec, Canada; 2Universite
´Laval, Faculty of Medicine, CHA, Enfant-Je
Hospital, Quebec, Canada; 3ALS Center, Department of Neurology, CHU Gui de Chauliac, Montpellier, France; 4Department of Pathology and Cellular Biology, Faculty of Medicine,
´al, Montreal, Quebec, Canada; 5Department of Medicine, Faculty of Medicine, Universite
´de Montreal, Montreal, Quebec, Canada and 6Research 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 2 bp 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 31 110 219 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 (425 bp) 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.9–11 But,
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 13.12 Unfortunately, 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.12 The 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
Isabelle Thibault and Pierre Provencher for sample collection and organization;
Anne Noreau, Cynthia Bourassa, Sophie Massart and Bertrand Boutie
technical support; and to acknowledge support from the Association pour la
Recherche sur la Scle
´rale Amyotrophique (ARS), the Association
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FUS splicing mutation in an ALS family
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