Analysis of FUS gene mutation in familial
amyotrophic lateral sclerosis within an
N. Ticozzi, MD
V. Silani, MD
A.L. LeClerc, BA
P. Keagle, BS
C. Gellera, PhD
A. Ratti, PhD
F. Taroni, MD
P.C. Sapp, BS
R.H. Brown, Jr., MD,
J.E. Landers, PhD
Objective: Mutations in the FUS gene on chromosome 16 have been recently discovered as a
cause of familial amyotrophic lateral sclerosis (FALS). This study determined the frequency and
identities of FUS gene mutations in a cohort of Italian patients with FALS.
Methods: We screened all 15 coding exons of FUS for mutations in 94 Italian patients with FALS.
Results: We identified 4 distinct missense mutations in 5 patients; 2 were novel. The mutations
were not present in 376 healthy Italian controls and thus are likely to be pathogenic.
Conclusions: Our results demonstrate that FUS mutations cause ?4% of familial amyotrophic
lateral sclerosis cases in the Italian population. Neurology®2009;73:1180–1185
ALS ? amyotrophic lateral sclerosis; FALS ? familial amyotrophic lateral sclerosis; FTD ? frontotemporal dementia; gDNA ?
genomic DNA; LMN ? lower motor neuron; NLS ? nuclear localization signal; UMN ? upper motor neuron.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of adult life characterized by
a selective loss of upper motor neurons (UMN) and lower motor neurons (LMN). The cause of
the disease is largely unknown. Approximately 10% of all ALS cases are familial (FALS), of
which ?20% are caused by mutations in the SOD1 gene.1Variants in TARDBP, which
encodes the DNA/RNA binding protein TDP-43, account for an additional ?5% of cases.2-4
Pathogenic mutations in 5 other genes (ALS2, SETX, VAPB, DNCT1, and ANG) have also
been associated with atypical ALS phenotypes in isolated families.5,6Recently, mutations in the
FUS gene, which also encodes a DNA/RNA binding protein, have been identified in ?5% of
patients with FALS who tested negative for SOD1 and TARDBP mutations.7,8The similarities
between TARDBP and FUS suggest that alterations in RNA processing may play an important
role in ALS pathogenesis. The FUS gene was initially isolated analyzing chromosomal translo-
cations in myxoid liposarcoma9and subsequently found in a variety of cancer-associated fusion
genes. The protein plays a role in transcription, splicing,10and shuttling of RNA from the
nucleus to the cytoplasm.11FUS also acts as a transcriptional regulatory sensor of DNA damage
signals and thus is required in maintaining the integrity of the genome.12FUS is diffusely
expressed in the CNS where it regulates the morphology of hippocampal dendritic spines and
stabilizes the structure of the synapse.13To further define the frequency and spectrum of FUS
mutations, we have screened a cohort of Italian patients with FALS.
e-Pub ahead of print on September 9, 2009, at www.neurology.org.
From the Department of Neurology (N.T., A.L.L., P.K., D.M.M.-Y., P.C.S., R.H.B., J.E.L.), University of Massachusetts Medical School, Worcester;
Department of Neurology and Laboratory of Neuroscience (N.T., V.S., A.R.), Centro “Dino Ferrari,” Universita ` degli Studi di Milano-IRCCS
Istituto Auxologico Italiano, Milan; Unit of Genetics of Neurodegenerative and Metabolic Diseases (C.G., F.T.), Fondazione IRCCS Istituto
Neurologico “Carlo Besta,” Milan, Italy; Department of Neurology (T.J.K.), Massachusetts General Hospital, Boston; and Howard Hughes Medical
Institute and Department of Biology (P.C.S.), Massachusetts Institute of Technology, Cambridge.
Supported by the ALS Therapy Alliance, Project ALS, the Angel Fund, the Pierre L. de Bourgknecht ALS Research Foundation, the Al-Athel ALS
Research Foundation, the ALS Family Charitable Foundation, and the National Institute of Neurological Disorders and Stroke (NS050557 and
NS050641). N.T., V.S., and A.R. were supported by the Italian Ministry of Health (Malattie Neurodegenerative, ex Art.56, n.533F/N1). F.T. and
C.G. were supported by the Italian Ministry of Health grant RF2007/INN644440. P.S. was supported by the Howard Hughes Medical Institute
(HHMI) through the auspices of Prof. H. Robert Horvitz, an Investigator in the HHMI. R.H.B. is a cofounder of AviTx, which targets development
Disclosure: Author disclosures are provided at the end of the article.
Editorial, page 1172
Address correspondence and
reprint requests to Dr. John E.
Landers, LRB604, 364 Plantation
St., Worcester, MA 01605
Copyright © 2009 by AAN Enterprises, Inc.
METHODS Patients and controls. Peripheral blood sam-
ples were collected from 94 unrelated patients with FALS (53
men and 41 women) in the period between 2002 and 2007. All
the patients were of Italian descent and were diagnosed with
probable or definite ALS according to the El Escorial revised
criteria.14Familial history was considered positive for ALS if the
proband had at least 1 affected relative within 3 generations. The
disease onset was bulbar in 24% of patients and spinal in 76%.
Six patients (4 men and 2 women) developed frontotemporal
dementia (FTD). In agreement with previous epidemiologic sur-
veys on motor neuron disease in Italy,15an initial involvement
of bulbar motor neurons was slightly more frequent in
women (30%) than men (19%). The average age at onset in
our cohort was 46.8 ? 15.2 years. Mutations in SOD1,
ANG,16and TARDBP17were excluded in all samples in the
present study. Control DNA was obtained from 376 unre-
lated, age-matched Italian subjects with a negative personal
and familial history for neurodegenerative diseases.
Standard protocol approvals, registrations, and patient
consents. We received approval for this study from the ethical
standards committees on human experimentation of the IRCCS
Istituto Auxologico Italiano and of the Fondazione IRCCS Isti-
tuto Neurologico “Carlo Besta.” Written informed consent was
obtained from all patients and healthy subjects participating in
the study (consent for research).
Molecular analysis. Genomic DNA (gDNA) was extracted
from peripheral blood leukocytes using standard procedures and
was subsequently amplified using the IllustraTMGenomiPhi HY
DNA Amplification Kit (GE Healthcare Life Sciences, Little Chal-
FUS were amplified by touchdown PCR using primers located in
adjacent intronic or noncoding regions. PCR primers and condi-
tions have been described elsewhere.7PCR products were subse-
quently purified by incubation with Exonuclease I and Shrimp
Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and
then resolved by capillary electrophoresis on an ABI 3730XL DNA
Analyzer (Applied Biosystems). Sequence analysis was per-
formed using the PHRED/PHRAP/Consed software suite
(www.phrap.org/phredphrapconsed.html),18-21and variations in
the sequences were identified with the Polyphred v6.15 software
mutations were further confirmed by bidirectional sequencing of
the original gDNA sample. The screening of the c.701G?T
mutation in control samples was performed using a custom Taq-
Man SNP Genotyping Assay using the File Builder 3.1 software
(Applied Biosystems) which consisted of a mix of unlabeled PCR
forward and reverse primers (forward: 5?-GCGGCGGTGGT-
GGTT-3?; reverse: 5?-CACGACCTCTGGGTTCATAGC-3?)
and 2 dye-labeled probes (allele X: VIC-CACTGCTGCGGT-
TGT-NFQ; allele Y: FAM-CACTGCTGAGGTTGT-NFQ).
Since the TaqMan assay designed for the c.467G?A mutation
failed to generate consistent results, exon 5 of the FUS gene was
directly sequenced in all control samples.
Bioinformatic analysis. The effect on splicing of the non-
coding and synonymous variants identified was assessed using
the electronic tools ESEfinder 3.0 (http://rulai.cshl.edu/tools/
html),25and SpliceView (http://bioinfo.itb.cnr.it/oriel/splice-
view.html).26In silico prediction of the putative functional ef-
fects of the missense mutations was obtained with the SNAP
RESULTS To identify novel FUS mutations and
further define the frequency of all FUS gene variants
in FALS, we sequenced the coding region (exons
1–15) and the intron/exon boundaries in 94 FALS
samples of Italian descent. Our sequence analysis
identified 2 previously described missense mutations
in the FUS gene. The mutation c.1561C?T pro-
duces an arginine to cysteine change at amino acid
521 (p.R521C) and was observed in 2 patients. The
c.1561C?G mutation was identified in a single pa-
tient and alters the same base pair but changes the
arginine residue to a glycine (p.R521G). In addition,
we have identified 2 novel missense mutations. The
mutation c.467G?A was identified in a single pa-
tient; it changes a glycine to a glutamic acid at posi-
tion 156 (p.G156E). The mutation c.701G?T, also
identified in a single patient, changes an arginine res-
idue to a leucine at position 234 (p.R234L). The
pedigrees of the patients carrying FUS mutations are
shown in figure 1. Chromatograms displaying the
mutations are shown in figure e-1 on the Neurology®
Web site at www.neurology.org. Unfortunately,
since all the affected relatives of the probands are
deceased, we could not evaluate the segregation of
the mutations in families. In addition to the missense
mutations, we also identified a novel synonymous
substitution (c.1566G?A [p.R522R]) as well as 5
novel variants in intronic (c.832 ? 36A?G,
c.833 ? 29C?T, c.1066 ? 82C?G) and 3? UTR
(c.*10C?T, c.*41G?A) regions of FUS (table e-1).
In silico analysis with the ESEfinder 3.0 software
showed that the synonymous variant does not dis-
rupt any exonic splicing enhancer motif, while
NNSplice and SpliceView programs predicted that
the intronic and 3? UTR variants do not affect splic-
ing mechanisms. These variants were not further
To determine whether the 2 novel missense muta-
tions are low allele frequency polymorphisms in the
population, we genotyped the p.G156E and
p.R234L mutations on a panel of 376 healthy con-
trol samples of Italian descent. Neither variant was
identified in these normal controls. This finding
strongly argues that these missense variants represent
The p.G156E is located within the SYQG do-
main of the FUS protein, which acts as a transcrip-
tional activation domain. Additionally, the p.R234L
mutation is located within the G-rich domain, a re-
gion in which 3 mutations have previously been
reported (figure 2). To further evaluate the pathoge-
nicity of these novel mutations, we used 2 software
Neurology 73October 13, 2009
applications, PMut and SNAP, which predict
whether a missense mutation is pathogenic or be-
nign. As expected, the previously described muta-
tions (p.R521C and p.R521G) were predicted to be
deleterious by both PMut and SNAP (table). The
novel mutations p.G156E and p.R234L are similarly
predicted to be deleterious by both PMut and SNAP
(table). In support of this prediction, we note that
the residues affected by these mutations (G156 and
R234) are highly conserved, being present in mam-
mals, reptiles, and zebra fish (figure 3). This observa-
tion also supports the view that the corresponding
missense mutations are adverse and likely to be
Phenotypic information for the 5 patients carry-
ing FUS mutations is provided in the table. Detailed
clinical records were available for 3 (B303, A51, and
B153) of the 5 patients and other affected family
members, and are reported in appendix e-1. Patient
B303 harbors the p.G156E mutation whereas both
A51 and B153 have the p.R521C mutation. Nota-
bly, each affected individual in the 3 pedigrees devel-
oped early symptoms of symmetric weakness of the
scapular or pelvic girdles and of the proximal muscles
of the upper or lower limbs. Also prominent was the
involvement of the axial muscles of the neck and
In total, we have identified 5 missense mutations
within a panel of 94 Italian patients with FALS
which were prescreened and found not to have muta-
tions in the SOD1 and TARDBP genes (which repre-
sent ?25% of all FALS cases). We therefore estimate
Figure 2Location of FUS mutations in familial amyotrophic lateral sclerosis
indicate the exons where mutations have been identified. (B) Linear protein structure of FUS indicating the different do-
mains. Amino acid changes are shown above the protein with arrows indicating their position. Mutations found in Italian
patients are shown in red.
Figure 1 Pedigrees of familial amyotrophic lateral sclerosis cases harboring FUS mutations
This illustrates the structure of the 5 unrelated Italian pedigrees to which the 5 cases studied here belong. Affected
individuals are shown in black; unaffected individuals are shown in white. Arrows indicate the individual harboring the
Neurology 73 October 13, 2009
that ?4% of FALS cases within the Italian popula-
tion are caused by mutation in the FUS gene. This
frequency is similar to that reported in a recent
screening in another FALS cohort.29
DISCUSSION FUS mutations were initially identi-
fied in 20 cases of FALS.7,8A vast majority of the
alterations (16 out of 20) were missense mutations
clustered in 5 arginine residues located in exon 15 of
the FUS gene. Interestingly, a p.H517Q homozy-
gous mutation within exon 15 was found in a family
presenting a recessive inheritance pattern; all other
identified families displayed a dominant inheritance
pattern. Additional mutations were identified in
exon 5 (p.174–175insGG, p.173–174delGG), exon
6 (p.R244C), and exon 14 (p.R514G). The pre-
dicted amino acid sequence of the FUS protein is
highly evolutionarily conserved and consists of an
N-terminal SYQG-rich region that acts as a tran-
scriptional activation domain, two G-rich regions, an
RNA binding domain, a Cys2/Cys2-zinc finger mo-
tif, and a C-terminal RGG-rich region, which con-
tains the nuclear localization signal (NLS).30,31
In our cohort of 94 unrelated Italian patients with
FALS, we identified FUS mutations in 5 patients.
The observed mutational frequency in our Italian co-
hort is similar to that observed in the 2 previous stud-
ies of FUS mutations in ALS.7,8Previous mutational
screenings have suggested that the prevalence of
SOD1 mutations in Italian ALS families is about
15%.32,33ANG mutations account for ?2% of
all familial cases,16while a recent study identified
TARDBP mutations in 6 out of 125 Italian patients
with FALS (4.8%).17To date, in the Italian popula-
tion, mutations in the ALS2 gene have been reported
only in an handful of families affected by infantile
ascending hereditary spastic paralysis34or juvenile
primary lateral sclerosis,35while a screening for
VAPB variants in patients with ALS was negative.36
To our knowledge, there are no reports of SETX or
dynactin mutations in Italian patients with ALS. Our
findings thus suggest that FUS mutations represent
the third most significant cause of FALS in Italy after
SOD1 and TARDBP.
In our cohort, we found 2 previously identified
mutations, p.R521G and p.R521C. The substitution
of the arginine at codon 521 disrupts the NLS and
leads to an aberrant subcellular distribution of FUS,
with retention and apparent aggregation of the mu-
tant protein in the cytoplasm.7The 2 novel muta-
tions observed in our cohort are located in the
N-terminal SYQG-rich domain (p.G156E) and in
the first G-rich region (p.R234L), where other muta-
tions have been found7(figure 2B). No data are avail-
able on the functional role of these variants, since
they are not located in the NLS and thus presumably
do not alter the subcellular localization of FUS.
Moreover, very little information is available on the
activity of both the SYQG-rich and G-rich domain
of FUS. We hypothesize, however, that p.G156E
and p.R234L are pathogenic for several reasons. The
amino acids at these positions are highly evolutionar-
ily conserved (figure 3) and these variants were not
detected in 376 Italian healthy controls (752 chro-
mosomes). Finally, both in silico SNAP and PMut
software analyses predicted that p.G156E and
p.R234L affect protein function (table), although
these data should be interpreted with caution.
Figure 3 Evolutionary conservation of FUS mutations
The evolutionary conservation of all 4 identified mutations is shown. For each, the mutated
amino acid is shown in red.
Table Phenotypic description of patients with FUS mutations
M c.467G?A G156E533 Bulbar11UMN/LMN YesPathogenic Non-neutral
F c.701G?TR234L6 66Spinal N/A N/ANoPathogenic Non-neutral
M c.1561C?TR521C 15 32Bulbar11 LMN No Pathogenic Non-neutral
F c.1561C?T R521C1525 Spinal60 LMNNo Pathogenic Non-neutral
F c.1561C?G R521G 15 n/aSpinal N/AN/ANoPathogenic Non-neutral
UMN/LMN ? presence of both upper and lower motor neuron signs; LMN ? prevalence of lower motor neuron signs; N/A ?
Neurology 73October 13, 2009
We note that the clinical presentation of the Ital-
ian patients with FUS gene mutations (symmetric,
proximal, and axial weakness at onset) was distinct
from typical ALS, which often begins in a single limb
distally.37Severe weakness of neck extensor muscles
at onset, as seen in one Italian case, is present in only
about 1% of patients with ALS,38and thus is also
unusual. We also note that in his fourth decade, pa-
tient B303 (p.G156E mutation) developed FTD
concurrently with motor neuron disease; patients
with ALS in the 2 previous reports on FUS muta-
tions were not described as having concomitant de-
mentia. It will be of interest to determine whether
FUS mutations consistently correlate with these dis-
tinctive clinical phenotypes in other patients with
Given the complexity of cellular functions and
pathways in which FUS is involved, further studies
will be required to delineate the explicit mechanisms
whereby these FUS mutations cause motor neuron
death. In particular, it will be of interest to assess
if the pathogenic pathways involving FUS and
TARDBP overlap. The 2 genes encode for DNA/
RNA binding proteins involved in gene regulation
and RNA processing. Mutations in FUS and TARDBP
may alter the binding of their nucleic acid targets,
resulting in altered mRNA splicing. Additionally,
some mutations have been shown to impair the capa-
bility of both proteins to shuttle between the nucleus
and the cytoplasm.39Widespread defects in RNA
processing as a result of either, or both, of these
mechanisms could lead to motor neuron death. The
precise pathogenic mechanism notwithstanding, our
study provides strong evidence that mutations in the
FUS gene represent a significant cause of familial
ALS in the Italian population. It also suggests that
some patients carrying FUS mutations may have un-
common clinical phenotypes, a possibility that mer-
its further analysis as more opportunities arise to
correlate FUS genotypes with clinical phenotypes.
Writing team: N.T., V.S., A.L., P.K, R.H.B., J.L.; all others received and
approved the manuscript. Patient selection, preliminary genetic screening,
and record collection: N.T., V.S., A.R., C.G., F.T., P.S. Sequencing and
genotyping: N.T., A.L., P.K. Data analysis: N.T., V.S., R.H.B., J.E.L.
Scientific planning and direction: N.T., V.S., A.L., T.K., R.H.B., J.E.L.
The authors thank the Italian patients with ALS and their caregivers and
the Peviani family for support.
Dr. Ticozzi has received research support from the Italian Ministry of
Health (Malattie Neurodegenerative, ex Art. 56, n533F/N1). Dr. Silani
serves on the editorial advisory boards of Amyotrophic Lateral Sclerosis,
European Neurology, and the Journal of Pediatric Neurology, and receives
research support from the Italian Ministry of Health (Malattie Neurode-
generative, ex Art. 56, n533F/N1). A.L. Leclerc and P. Keagle report no
disclosures. Dr. Gellera has received research support from the Italian
Ministry of Health (#RF2007/INN644440). Dr. Ratti has received re-
search support from the Italian Ministry of Health (Malattie Neurodegen-
erative, ex Art. 56, n533F/N1). Dr. Taroni serves on the scientific
advisory board of the Italian Ataxia Association (AISA); serves on the
editorial board of the Journal of Neurology; and receives research support
from ApoPharma Inc. [EudraCT No. 2007-003331-23 and No. 2009-
010865-22 (Unit PI)], the Telethon Foundation (Telethon and
Telethon-UILDM Project Coordinator), and the Italian Ministry of
Health [AIFA EudraCT No. 2007-003357-85 (PI) and MoH RF2007/
INN644440)]. Dr. Kwiatkowski has served as consultant and received
funding for international travel from Asia Ventures Management; receives
license fee payments as a contributor to US Patents 5,741,645 (issued:
1998) and 5,834,183 (issued: 1998) entitled “Gene sequence for spino-
cerebellar ataxia type 1 and method for diagnosis” and may accrue revenue
from US patent 61/135,689, “Genetic test for FUS ALS” (filed: 2008);
has served as consultant and received honoraria from Athena Diagnostics,
Inc.; and receives research support from the NIH [R01NS050557 (Co-PI)]
and from the ALS Therapy Alliance, the Angel Fund and Project ALS,
the Pierre L. de Bourgknecht ALS Research Foundation, the Al-Athel
ALS Research Foundation, and the ALS Family Charitable Foundation.
D.M. McKenna-Yasek receives salary support from the NIH [RO1-
NS050641-04]. P.C. Sapp has received research support from the
Howard Hughes Medical Institute (grant awarded to Prof. H. Robert
Horvitz). Dr. Brown serves on scientific advisory boards for Biogen Idec,
Acceleron Pharma, Link Medicine, and AviTx Inc.; has filed/holds US
Patent 5,843,641, “Methods for the diagnosis of familial amyotrophic
lateral sclerosis” (filed: 1993); receives royalties from publishing Principles
of Neurology (McGraw-Hill, 2005); receives research support from the
NIH [NINDS R01NS050557 (PI) and NINDS UO1NS052225-02
(PI)], the ALS Therapy Alliance, Project ALS, the Angel Fund, the Pierre
L. de Bourgknecht ALS Research Foundation, the Al-Athel ALS Research
Foundation, and the ALS Family Charitable Foundation; receives board
of directors compensation from AviTx Inc. (co-founder); and receives
license fee payments from Athena Diagnostics, Inc. related to diagnostic
blood tests. Dr. Landers has received payments from MIT as an inventor
of US Patent 6,703,228; has been deposed concerning a lawsuit between
Affymetrix, Inc. and E8/MIT regarding this patent; and has received re-
search support from the ALS Therapy Alliance, Project ALS, the Angel
Fund, the Pierre L. de Bourgknecht ALS Research Foundation, the Al-
Athel ALS Research Foundation, and the ALS Family Charitable Founda-
tion. His spouse is an employee of Bristol-Myers Squibb.
Received March 19, 2009. Accepted in final form July 1, 2009.
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