Genetic contribution of FUS to

frontotemporal lobar degeneration

T. Van Langenhove, MD
J. van der Zee, PhD
K. Sleegers, MD, PhD
S. Engelborghs, MD,
PhD
R. Vandenberghe, MD,
PhD
I. Gijselinck, PhD
M. Van den Broeck, BSc
M. Mattheijssens, BSc
K. Peeters, BSc
P.P. De Deyn, MD,
PhD
M. Cruts, PhD
C. Van Broeckhoven,
PhD, DSc

ABSTRACT

Background: Recently, the FUS gene was identified as a new causal gene for amyotrophic lateral
sclerosis (ALS) in 4% of patients with familial ALS. Since ALS and frontotemporal lobar degeneration (FTLD) are part of a clinical, pathologic, and genetic disease spectrum, we investigated a
potential role of FUS in FTLD.

Methods: We performed mutational analysis of FUS in 122 patients with FTLD and 15 patients
with FTLD-ALS, as well as in 47 patients with ALS. Mutation screening was performed by sequencing of PCR amplicons of the 15 FUS exons.
Results: We identified 1 patient with FTLD with a novel missense mutation, M254V, that was
absent in 638 control individuals. In silico analysis predicted this amino acid substitution to be
pathogenic. The patient did not have a proven family history of neurodegenerative brain disease.
Further, we observed the known R521H mutation in 1 patient with ALS. No FUS mutations were
detected in the patients with FTLD-ALS. While insertions/deletions of 2 glycines (G) were suggested to be pathogenic in the initial FUS reports, we observed an identical GG-deletion in 2
healthy individuals and similar G-insertions/deletions in 4 other control individuals, suggesting
that G-insertions/deletions within this G-rich region may be tolerated.

Conclusions: In a first analysis of FUS in patients with frontotemporal lobar degeneration (FTLD),
Address correspondence and
reprint requests to Prof. Dr.
Christine Van Broeckhoven,
VIBDepartment of Molecular
Genetics, Neurodegenerative
Brain Diseases Group, University
of AntwerpCDE,
Universiteitsplein 1, B-2610
Antwerpen, Belgium
christine.vanbroeckhoven@molgen.vib-ua.be

Editorial, page 354

we identified a novel FUS missense mutation, M254V, in 1 patient with pure FTLD. At this point,
the biologic relevance of this mutation remains elusive. Screening of additional FTLD patient
cohorts will be needed to further elucidate the contribution of FUS mutations to FTLD
pathogenesis. Neurology 2010;74:366 371
GLOSSARY
ALS amyotrophic lateral sclerosis; FTD frontotemporal dementia; FTLD frontotemporal lobar degeneration; TDP
TAR DNA-binding protein.

Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are 2
related, ultimately fatal neurodegenerative disorders. FTLD is a focal dementia syndrome affecting primarily the frontal and temporal lobes of the brain whereas ALS is predominantly a
disease of the lower and upper motor neurons. Although distinct identities, accumulating
evidence supports the view that both diseases are part of a clinical, pathologic, and genetic
spectrum. In up to 50% of patients with ALS, mild disturbances of executive functions can be
observed related to frontal lobe dysfunction, and in 10% cognitive and behavioral changes
are present that meet the criteria of FTLD.1,2 Conversely, a proportion of patients with FTLD
will develop muscle wasting and spasticity in later stages of the disease.3 At neuropathologic

Supplemental data at
www.neurology.org
From the Neurodegenerative Brain Diseases Group (T.V.L., J.v.d.Z., K.S., I.G., M.V.d.B., M.M., K.P., M.C., C.V.B.), Department of Molecular
Genetics, VIB, Antwerpen; Laboratory of Neurogenetics (T.V.L., J.v.d.Z., K.S., I.G., M.V.d.B., M.M., K.P., M.C., C.V.B.) and Laboratory of
Neurochemistry and Behavior (S.E., P.P.D.D.), Institute Born-Bunge, Antwerpen; University of Antwerp (T.V.L., J.v.d.Z., K.S., S.E., I.G.,
M.V.d.B., M.M., K.P., P.P.D.D., M.C., C.V.B.), Antwerpen; Memory Clinic and Division of Neurology (S.E., P.P.D.D.), ZNA Middelheim,
Antwerpen; and Department of Neurology (R.V.), University Hospitals Leuven and University of Leuven, Leuven, Belgium.
Study funding: This study was partly funded by the Interuniversity Attraction Poles (IAP) program P6/43 of the Belgian Science Policy office, the
Foundation for Alzheimer Research (SAO/FRMA), a Methusalem excellence grant of the Flemish Government, the Fund for Scientific
ResearchFlanders (FWO-V), the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-V), the Special
Research Fund of the University of Antwerp, and the Katholieke Universiteit Leuven, Belgium. T.V.L. is holder of a PhD fellowship of the IWT-V.
The FWO-V provided a postdoctoral fellowship to J.v.d.Z. and K.S.
Disclosure: Author disclosures are provided at the end of the article.
366

Copyright 2010 by AAN Enterprises, Inc.

examination, over half of FTLD patients

brains show pathologic aggregates of ubiquitinated TAR DNA-binding protein 43 (TDP43) in cortical neurons of affected regions
(FTLD-TDP).4-6 Similarly, for the majority
(90%) of patients with ALS, inclusions of
TDP-43 were detected in degenerating motor
neurons.5,7 The link between FTLD and ALS
is also supported by genetic evidence. Several
families have been reported in which affected
family members manifest FTLD, ALS, or
both, and showed linkage to a region on chromosome 9p13-21.8-10
Recently a new causal gene was identified for
ALS. Mutations in the Fused in Sarcoma gene
(FUS) were found segregating with ALS in families linked to chromosome 16. Two independent articles described a total of 15 different
FUS mutations in 26 unrelated ALS families
(4% of familial ALS).11,12 Twelve were missense mutations, clustering in the C-terminal region of the protein encoded by exons 14 and 15.
In addition, 1 missense mutation and a deletion
and insertion of 2 glycine (G) residues were
identified in the upstream G-rich region of FUS
in exons 5 and 6 (figure). The clinical presentation of FUS mutations was classic ALS, with a
mean age at onset of 46 years and mean disease
duration of 33 months.
The FUS protein shows remarkable functional and pathologic similarities with TDP43.13,14 Both FUS and TDP-43 are nuclear
proteins involved in RNA and DNA metabolism. The neuropathologic examination of
FUS mutation carriers revealed inclusions of
FUS protein within the cytoplasm of affected
motor neurons. Cellular expression studies
further showed subcellular mislocalization
and cytoplasmic retention of mutant FUS
protein.11,12 These observations are similar to
the aberrant TDP-43 processing seen in
TDP-43 proteinopathies.5,6 Following the
identification of TDP-43 as a major pathologic protein in both ALS and FTLD, pathogenic mutations were detected in the
encoding TARDBP gene in patients with
ALS,15,16 establishing a primary genetic role
for TARDBP in ALS. Whereas the initial mutation screens of TARDBP in FTLD were
negative,17,18 rare TARDBP variations have

now also been identified in patients with

FTLD-ALS and FTLD in the absence of
ALS19-21 (AD & FTD Mutation Database,
http://www.molgen.ua.ac.be/FTDMutations).
The discovery of FUS as an important disease gene in ALS prompted us to investigate a
possible role for FUS in FTLD and within the
FTLD-ALS spectrum. We set up a mutation
screen of FUS in a series of patients with
FTLD (n 122), ALS (n 47), and concurrent FTLD-ALS (n 15).
METHODS Study population. Included in this study were
122 patients with FTLD, 47 patients with ALS, and 15 patients
with FTLD-ALS. The patients were ascertained between 1998
and 2009 within the framework of 2 ongoing prospective clinical
studies of dementia conducted at the memory clinics and neurology departments of the ZNA Middelheim, Antwerpen, and the
University Hospitals, Leuven, in Belgium. All patients were evaluated following a standard protocol including neurologic examination, neuropsychological testing, biochemical analysis, EEG,
and neuroimaging.22 Clinical diagnosis was reached in consensus
by 2 neurologists according to the Lund and Manchester group
criteria for FTLD3 and the revised El Escorial criteria for ALS.23
In the ALS study population, additional patients were included
who had initially been referred to our Molecular Diagnostic Unit
for genetic testing. Of the 122 patients with FTLD, 96 were
diagnosed with frontotemporal dementia (FTD), 11 with progressive nonfluent aphasia, and 10 with semantic dementia. For
5 patients, the FTLD subtype remained unspecified. Seven patients with FTLD were diagnosed pathologically with FTLDTDP, 1 with FTLD-tau, 2 with FTLD-U, 2 with FTLD-UPS,
and 1 with FTLD-NI, conforming with the new consensus recommendations for nomenclature of FTLD neuropathologic subtypes.24 One patient with FTLD-ALS came to autopsy with
FTLD-TDP pathology. Previous mutation analyses of the
known FTLD genesMAPT, PGRN, PSEN1, VCP, and
CHMP2Bidentified 12 mutations (10%) in the patients with
FTLD, and screening of the known ALS genes SOD1, ANG, and
TARDBPidentified 4 mutations (9%) in the patients with
ALS. In the FTLD-ALS patient group, no mutations in the
FTLD or ALS genes were observed. As control population, we
included 638 neurologically healthy community individuals.
Descriptions of patient and control study populations are summarized in table 1.

Standard protocol approvals, registrations, and patient

informed consents. All patients or their legal guardians gave
written informed consent for participation in both the clinical
and genetic studies. The clinical study protocol and the informed consent forms for patient ascertainment were approved
by the Medical Ethics Committee of ZNA Middelheim, and
University Hospitals Leuven, Belgium. The genetic study protocol and informed consent forms were approved by the Medical
Ethics Committee of the University of Antwerp, Belgium. Patient relatives and community-dwelling control individuals were
ascertained after written informed consent, within the frame of
the medical ethical approved genetic study protocol. All samples
received a unique identifier number and demographic, medical,
and genetic data were stored in a centralized database. Restrictive
Neurology 74

February 2, 2010

367

Table 1

the programs FSPLICE (http://www.softberry.com), NetGene2,27 and NNSplice.28

Characteristics of patient and control study populations

FTLD
(n 122)

ALS
(n 47)

FTLD-ALS
(n 15)

Controls
(n 638)

Mean age O/I SD, y

64.2 11.2

46.3 13.9

60.9 11.1

62.1 15.5

Male, n (%)

63 (51)

32 (68)

7 (46)

273 (43)

Familial, n (%)

36 (29)

18 (38)

7 (46)

No. mutations, n (%)

12 (10)a

4 (9)b

0 (0)

Abbreviations: ALS amyotrophic lateral sclerosis; FTLD frontotemporal lobar degeneration; O/I onset/inclusion.
a
Three PGRN IVS15GC, PGRN A89fsX41, PGRN M1I, PGRN genomic deletion, PGRN
R432C; MAPT G273R, MAPT S305, MAPT V363I; VCP R159H; PSEN1 G183V.36-40
b
SOD1 L38V, SOD1 L106V, SOD1 L144F, SOD1 C146X.

access permissions were given to researchers and clinicians depending on their respective role in this study.

FUS sequencing. All 15 FUS exons including intron-exon

boundaries were sequenced. Total genomic DNA of patients and
control individuals was prepared from peripheral blood or lymphoblast cell lines. Genomic DNA was PCR amplified using
primers designed by ExonPrimer as available trough the UCSC
Genome Browser (table e-1 on the Neurology Web site at
www.neurology.org). PCR amplicons were purified using the
ExoSAP-IT kit (USB Corporation, Cleveland, OH) and sequenced in both directions using the BigDye Terminator Cycle
Sequencing kit v3.1 (Applied Biosystems, Foster City, CA) on
an ABI3730 automated sequencer (Applied Biosystems). Sequence variations were detected using the software package novoSNP25 and confirmed by visual inspection of the DNA
sequence traces.

In silico sequence variant analysis. PMUT26 was used to

predict the impact of an amino acid substitution on the function
of the protein. The effect of sequence variants on splice site function and the prediction of alternative splice sites were assessed by

Figure

We performed a systematic mutation

analysis of all FUS exons on genomic DNA of 122
patients with FTLD and 15 patients with FTLDALS. In addition, we screened 47 patients with pure
ALS. Two patient-specific missense mutations were
detected, 1 in a patient with FTLD and 1 in a patient
with ALS (figure and table 2). Further, our analyses
identified in 2 neurologically healthy control individuals a GG deletion which was previously reported as
pathogenic.

RESULTS

FUS M254V in a patient with FTLD. A single nucleotide change in exon 6, c.760AG, was identified in a
patient with FTLD predicting a methionine to valine
substitution at codon 254 (M254V). We further sequenced exon 6 in 638 healthy elderly control individuals (mean age at inclusion 62.1 years; SD 15.5
years). This analysis did not identify any individual
carrying M254V, suggesting that the M254V mutation is associated with FTLD. Multiple alignments of
FUS homologues of different species indicated that
M254V altered a highly conserved amino acid residue (figure). In addition, alignment of sequences of
Ewing sarcoma breakpoint region 1 protein (EWS),
which belongs together with FUS to the RRM TET
family,29 also revealed conservation of residue M254,
further underpinning its functional relevance (data
not shown). In silico analysis by PMUT, a software
program that allows the prediction of the pathogenic

FUS mutations in amyotrophic lateral sclerosis and frontotemporal lobar degeneration

(A) Schematic representation of reported FUS mutations relative to the gene structure and the proteins functional domains. Mutations identified in the
present study are highlighted in bold. QGSY-rich glutamine, glycine, serine, tyrosinerich region; G-rich glycine-rich region; NES nuclear export
signal; RRM RNA recognition motifs; RGG-rich arginine, glycinerich region; Zn F zinc finger. (B) Sequence chromatograms of FUS mutations
identified in a Belgian patient with frontotemporal lobar degeneration (c.760AG, M254V) and a Belgian patient with amyotrophic lateral sclerosis
(c.1562GA, R521H) and alignment of FUS homologues displaying evolutionary conservation of residues M254 and R521 across species.
368

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Table 2

Clinical features of FUS mutation carriers

Mutation

Diagnosis

Age at
onset

M254V

FTD

R521H

ALS

Cognitive deficits

Motor symptoms

PET

EMG

52 y

Progressive behavior and

personality changes,
minor amnesia

Minor extrapyramidal rigidity

Decreased tracer uptake

frontal and temporal,
right left

NA

33 y

Normal

Progressive asymmetric lower

motor neuron loss in limbs,
legs affected before arms

NA

Signs of active denervation

in 4 limbs, normal sensory
nerve action potential

Abbreviations: ALS amyotrophic lateral sclerosis; FTD frontotemporal dementia; NA not assessed.

effect of missense mutations based on protein conservation and conformation,26 indicated that M254V is
most likely pathogenic (output 0.622; 0.5 predicts a pathologic mutation). Previous mutation
analyses of MAPT, PGRN, VCP, and CHMP2B in
this patient excluded causal mutations.
The patient displayed first aberrant behavior and
personality changes at the age of 52 years. An FDGPET scan confirmed the diagnosis of FTLD with decreased tracer uptake bilaterally frontal and temporal,
most pronounced at the right side. At her last medical examination, at age 55 years, there were no clinical signs of lower motor neuron disease. By that time,
the patient had developed slight extrapyramidal rigidity. Inquiry for family history of neurodegenerative brain diseases revealed 1 maternal uncle who had
developed dementia at a later age.
FUS R521H in a patient with ALS. A second nonsyn-

onymous substitution, arginine to histidine at residue 521 (R521H), was identified in a patient with
ALS. This mutation had already previously been
linked to ALS in multiple affected members of 3
families and was absent in 1,846 control individuals.11,12 We additionally excluded this variation in
180 control individuals. No pathogenic mutations
had been detected in this patient in the SOD1,
TARDBP, and ANG genes.
The patient experienced first symptoms of pain
and fatigue in the left leg at age 33 years. Her symptoms progressed rapidly over a period of 5 months to
involve paresis of left and right limbs and difficulties
with swallowing. Needle EMG testing at this time
revealed signs of active denervation in all 4 limbs,
most pronounced in the 2 legs. Family history was
indicative of autosomal dominant inheritance: her
mother was diagnosed with ALS at age 60 years, her
grandmother at age 50 years, and 2 more siblings of
her mother were reported to have ALS.
G-insertions/deletions in the FUS G-rich region.

Screening of FUS exons 5 and 6 in 638 control individuals revealed a GG deletion in exon 5 in the G-rich
region of FUS (G174_G175delGG; figure, table e-2).
This mutation was previously suggested to be pathogenic in patients with ALS. At the gDNA level, the GG

deletion presents as a 6 base pair deletion located near

the exon 5 splice donor site. Prediction of splice sites
(FSPLICE, http://www.softberry.com, NetGene227
and NNSplice28) revealed with high reliability the use of
an alternative donor site, 6 base pairs upstream leading
to the deletion of 2 glycines. This GG deletion was
present in 2/638 control individuals, aged 70 and 37
years. An additional GGG-deletion and a G-insertion
in the G-rich region of FUS were also observed in 3
control individuals and 1 control individual, respectively (table e-2).
Recently, a new player was introduced in the FTLD-ALS disease spectrum with the
identification of FUS mutations in patients with familial ALS.11,12 To determine whether this newly
identified ALS gene could have a role in FTLD etiology, we performed a genetic mutation screen of FUS
in 122 patients with FTLD and 15 patients with
FTLD and ALS.
We detected 1 patient with FTLD with a novel
FUS missense mutation, M254V, located in exon 6
of FUS. The patient was diagnosed with behavioral
variant FTD and further clinical follow-up showed
no signs of lower motor neuron disease. In the initial
FUS reports, the majority of the missense mutations
(12/13) were clustering in the C-terminus of the protein except for 1 in exon 6, R244C. Therefore, the
question arises whether these rarer exon 6 variants
have biologic relevance to the disease. There is, however, genetic evidence that supports a potential
pathogenic role for M254V. First, this mutation was
absent in a 638 control individuals (mutant allele
frequency 0.078%). Second, multiple alignments
of FUS homologues and paralogues showed that the
M-residue at codon 254 is strongly conserved
throughout evolution. Third, in silico analysis predicted a likely pathogenic effect of M254V on FUS
functioning. Finally, mutations in known FTLD
genesMAPT, PGRN, VCP, and CHMP2Bwere
excluded in the FUS M254V mutation carrier. On
the other hand, the family history data that could be
collected from this patient were not compelling. One
maternal uncle developed dementia at a later age;
DISCUSSION

Neurology 74

February 2, 2010

369

however, both parents are currently still alive and

asymptomatic. DNA of the parents for mutation
testing could not be obtained. If pathogenic, this
mutation must either have arisen de novo or is not
fully penetrant. Reduced penetrance was demonstrated in an ALS family segregating FUS R521G.11
Possibly, some FUS mutations act as a susceptibility
factor rather than a fully penetrant mutation in developing neurodegenerative disease, including
FTLD.30,31 Immunohistochemistry will be needed to
allow a definite classification of the brain pathology
in this patient with FTLD and will provide more
insight in the biologic relevance of FUS M254V.
We also included 47 patients with a classic ALS
phenotype in the mutational analysis, of which 18
patients had a positive family history, and identified
the known R521H mutation in 1 familial ALS patient (1/18 or 5.6%). Familial history was conforming to an autosomal dominant inheritance of the
disease. We noted variable ages of onset of 33, 50,
and 60 years, indicating that other genetic or environmental modifying factors could be implicated in
the disease development. There was no indication of
cognitive deficits in any of the patients in this family.
In the group of patients with FTLD-ALS (n 15)
we did not find any mutations in FUS, although admittedly the study population was small.
The majority of FUS mutations linked to ALS
were missense mutations, except for 2 insertion/
deletion mutations in the G-rich region of FUS.11
The authors found a GG deletion in 3 related patients with ALS as well as a GG insertion within the
same glycine stretch in 1 patient with familial ALS.
The GG insertion and deletion were excluded in 176
control individuals. In our study, however, sequencing
of 638 control individuals revealed the GG deletion in 2
healthy individuals without clinical evidence of neurodegenerative disease. These GG-insertions/deletions are
located in a G10-stretch of the G-rich region of FUS, a
region that is not strongly conserved in homologues of
closely related organisms. Moreover, we observed similar insertions/deletions of G-residues in the G-rich domain in 4 other control individuals. Together, our
findings suggest that small G-insertions/deletions in this
region may well be tolerated.
At this point it is difficult to estimate the exact
contribution of FUS in FTLD genetic etiology, as
uncertainty still exists about the true pathogenicity of
M254V identified here. As more FTLD cohorts will
be analyzed, the impact of FUS in FTLD will become clearer. Considering the structural and functional parallel that exists between TDP-43 and
FUS,13,14 it is noteworthy that although TDP-43 inclusions are a major pathologic hallmark in patients
with ALS and patients with FTLD,4-7 dominant mu370

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February 2, 2010

tations in TARDBP are primarily observed in pure

ALS at one end of the FTLD-ALS spectrum.15-18 Recently, a few publications reported that the phenotype of TARDBP mutations may occasionally also
encompass FTLD.19-21 The same might possibly hold
for FUS. It will be interesting to see if the fraction of
patients with FTLD with ubiquitin-positive but
TDP-43-negative brain pathology (denoted FTLDUPS and about 10%20% of FTLD-U32-35) will display similar FUS neuropathology as observed in the
FUS-positive patients with ALS. Our series included
2 patients with FTLD-UPS pathology but they were
negative for FUS mutations.
ACKNOWLEDGMENT
The authors thank the patients for their cooperation in this study, the
personnel of the Genetic Service Facility of the VIBDepartment of Molecular Genetics (http://www.vibgeneticservicefacility.be), and the Antwerp Biobank of the Institute Born-Bunge (IBB).

DISCLOSURE
Dr. Van Langenhove, Dr. van der Zee, and Dr. Sleegers report no disclosures. Dr. Engelborghs serves on a scientific advisory board of JanssenCilag and UCB; and serves on the editorial advisory boards of Clinical
Neurology and Neurosurgery and Current Medical LiteratureNeurology.
Dr. Vandenberghe serves on the editorial board of Frontiers in Neuroscience and receives research support from GE Healthcare, Wyeth, Pfizer
Inc., Eli Lilly and Company, and Medivation, Inc. Dr. Gijselinck, M.
Van den Broeck, M. Mattheijssens, K. Peeters, Dr. De Deyn, Dr. Cruts,
and Dr. Van Broeckhoven report no disclosures.

Received July 15, 2009. Accepted in final form October 26, 2009.
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