Novel Mutations in TARDBP (TDP-43) in Patients with
Familial Amyotrophic Lateral Sclerosis
Nicola J. Rutherford1, Yong-Jie Zhang1, Matt Baker1, Jennifer M. Gass1, NiCole A. Finch1, Ya-Fei Xu1,
Heather Stewart2, Brendan J. Kelley3, Karen Kuntz3, Richard J. P. Crook1, Jemeen Sreedharan4,5, Caroline
Vance4,5, Eric Sorenson3, Carol Lippa6, Eileen H. Bigio7, Daniel H. Geschwind8, David S. Knopman3,
Hiroshi Mitsumoto9, Ronald C. Petersen3, Neil R. Cashman10, Mike Hutton1¤, Christopher E. Shaw4,5,
Kevin B. Boylan11, Bradley Boeve3, Neill R. Graff-Radford11, Zbigniew K. Wszolek11, Richard J. Caselli12,
Dennis W. Dickson1, Ian R. Mackenzie13, Leonard Petrucelli1, Rosa Rademakers1*
1Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, United States of America, 2The ALS Centre, Vancouver General Hospital, Vancouver, British Columbia,
Canada, 3Department of Neurology, Mayo Clinic, Rochester, Minnesota, United States of America, 4Department of Clinical Neuroscience, Medical Research Council (MRC)
Centre for Neurodegeneration Research, King’s College London, London, United Kingdom, 5Institute of Psychiatry, King’s College London, London, United Kingdom,
6Department of Neurology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America, 7Alzheimer Disease Center, Northwestern
University Feinberg School of Medicine, Chicago, Illinois, United States of America, 8Neurogenetics Program, Department of Neurology, The David Geffen School of
Medicine at University of California, Los Angeles, California, United States of America, 9Eleanor and Lou Gehrig MDA/ALS Research Center, Columbia University, New York,
New York, United States of America, 10Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada, 11Department of Neurology, Mayo
Clinic, Jacksonville Florida, United States of America, 12Department of Neurology, Mayo Clinic, Scottsdale, Arizona, United States of America, 13Department of
Pathology, University of British Columbia, Vancouver, British Columbia, Canada
The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis
(ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegen-
erative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were
recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this
study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we
identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases
associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V,
p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in
24 patients with sporadic ALS or 180 patients with other TDP-43–positive neurodegenerative diseases. The presence of
p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations
affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein
interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved
fragments, including the ,25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a
cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal
fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or
intracellular accumulation of TDP-43 fragments leading to apoptosis.
Citation: Rutherford NJ, Zhang Y-J, Baker M, Gass JM, Finch NA, et al. (2008) Novel Mutations in TARDBP (TDP-43) in Patients with Familial Amyotrophic Lateral
Sclerosis. PLoS Genet 4(9): e1000193. doi:10.1371/journal.pgen.1000193
Editor: Gregory A. Cox, The Jackson Laboratory, United States of America
Received April 21, 2008; Accepted August 7, 2008; Published September 19, 2008
Copyright: ? 2008 Rutherford et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH funding: Mayo Clinic ADRC (P50 AG16574), Mayo Clinic ADPR (U01 AG06786), R01 NS42759, R01 NS42759-04S1, P30
AG13854, AG19610-01, P50 AG25711, P01 AG17216, P50 NS 40256, P01 AG03949, R01 AG15866, R01 AG026251-01 and the Morris K. Udall NIH/NINDS PD Center
of Excellence award (NS40256). In addition, the authors received support from the Pacific Alzheimer’s Research Foundation (PARF) # C06-01, Canadian Institutes
of Health Research grant #74580, The Potamkin Foundation, The Justice Newmann Fund, The Robert H. and Clarice Smith and Abigail Van Buren Alzheimer’s
Disease Research Program, The M. L. Simpson Foundation Trust, Canada Research Chairs, PrioNet Canada, Amorfix Life Sciences, Cure PSP/Society for PSP, The
State of Florida Department of Elder Affairs and the Mayo Foundation. Sponsors and funders had no role in study design, data collection, analysis or
interpretation, or manuscript preparation, review or approval.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Rademakers.Rosa@mayo.edu
¤ Current address: Neuroscience Drug Discovery, Merck Research Laboratories, Boston, Massachusetts, United States of America
Transactive response DNA binding protein with a molecular
weight of 43 kDa (TDP-43) is a ubiquitously expressed nuclear
protein encoded by the TARDBP gene, located on chromosome
1p36. TDP-43 was identified as the major disease accumulated
protein in ubiquitinated neuronal cytoplasmic (NCI) and neuronal
intranuclear inclusions (NII), that define a growing class of
neurological diseases, collectively referred to as TDP-43 proteino-
pathies [1–5]. These diseases include amyotrophic lateral sclerosis
(ALS), frontotemporal lobar degeneration (FTLD) with ubiquitin
immunoreactive, tau negative inclusions (FTLD-U) and FTLD
PLoS Genetics | www.plosgenetics.org1September 2008 | Volume 4 | Issue 9 | e1000193
with motor neuron disease (FTLD-MND). In TDP-43 proteino-
pathies, TDP-43 is relocated from the nucleus to the cytoplasm
and sequestered into inclusions that are mainly composed of
hyperphosphorylated and C-terminally truncated TDP-43 frag-
ments [4,6,7]. TDP-43 immunoreactive histopathology has also
been reported in 20–30% of patients with Alzheimer’s disease
(AD), 70% of patients with hippocampal sclerosis (HpScl), 33% of
patients with Pick’s disease and in a subset of patients with Lewy-
body related diseases [8–12]. TDP-43 is a highly conserved
protein, containing 2 RNA recognition motifs and a C-terminal
glycine-rich domain, known to promote protein-protein interac-
TDP-43 can bind to the common microsatellite region (GU/
GT)nin RNA and DNA, with proposed functions in transcrip-
tional regulation . Most recent research has focused on the
role of TDP-43 in the regulation of exon 9 alternative splicing in
the cystic fibrosis transmembrane conductance regulator gene,
however, additional targets have been identified and others likely
await identification [14,15]. TDP-43 has also been implicated in
microRNA biogenesis .
ALS and FTLD-U are etiologically complex disorders with
genetic as well as environmental factors contributing to the
disease. A positive family history is reported in 5–10% of ALS
patients and in up to 50% of FTLD-U patients, often with an
autosomal dominant pattern of inheritance [17–19]. Mutations in
the Cu/Zn superoxide dismutase gene (SOD1) account for ,10–
20% of familial and 1–2% of apparent sporadic ALS patients .
However, TDP-43 inclusions were not present in SOD1 mutation
carriers, suggesting a distinct disease mechanism in these patients
. The genetic basis of FTLD-U is just starting to be understood
. Loss-of-function mutations in the gene encoding the secreted
growth factor progranulin (PGRN) are a major known cause of
familial FTLD-U [22,23], explaining up to 25% of patients
worldwide . Other rare genetic causes of familial FTLD-U
include mutations in the valosin containing protein gene (VCP) and
the gene encoding the charged multivesicular body protein 2B
(CHMP2B), while some families with a combination of FTLD and
ALS show genetic linkage to a locus on chromosome 9p [25–29].
Since rare missense mutations and multiplications have been
identified in genes encoding the major constituents of the
pathological deposits in several neurodegenerative diseases, we
hypothesized that mutations in TARDBP may contribute to the
development of TDP-43 proteinopathies. In fact, the first missense
mutations in TARDBP were recently discovered in 2 autosomal
dominant ALS families and 2 sporadic ALS patients, supporting
the central role for TDP-43 in disease pathogenesis [30,31]. A
large population-based study further identified 8 different missense
mutations in 3 familial and 6 sporadic ALS patients and showed
accumulation of a detergent-insoluble TDP-43 protein product of
,28 kDa . Here, we report on the extensive mutation
screening of TARDBP in a diverse cohort of clinical and
pathological confirmed patients with neurodegenerative diseases
characterized by TDP-43 pathology, which led to the identifica-
tion of 3 additional ALS families with TARDBP mutations. We
further show accumulation of proteolytic cleaved fragments with a
molecular weight of approximately 35 and 25 kDa in lympho-
blastoid cell lines derived from TARDBP mutation carriers.
TARDBP Mutation Analyses
We performed in silico analyses of the TARDBP gene structure
by alignment of human spliced expressed sequence tags listed in
the UCSC genome browser (http://genome.ucsc.edu/). This led
to the identification of a novel 59 non-coding exon (exon 0) in
addition to the known non-coding exon 1 and the 5 coding exons
that are included in the TARDBP reference mRNA sequence
(NCBI accession number NM_007375). Sequencing analyses of
the 5 coding and 2 non-coding exons of TARDBP in our initial
cohort of 176 clinical patients and 120 patients with pathologically
confirmed TDP-43 pathology revealed 3 heterozygous missense
mutations in 3 of the 116 analyzed ALS patients (2.6%), while no
mutations were detected in 180 patients affected with FTLD-U,
FTLD-MND, AD, HpScl and Lewy-body disease (Table 1,
Figure 1). Since all mutation carriers were index patients of
autosomal dominant ALS families, the frequency of TARDBP
mutations increased to 3.3% in the subpopulation of familial ALS
patients (3/92 patients). One silent mutation (p.Ala66) and 18
additional sequence variants in intronic and non-coding regions
The abnormal accumulation of disease proteins in neuronal
cells of the brain is a characteristic feature of many
neurodegenerative diseases. Rare mutations in the genes
that encode the accumulating proteins have been identi-
fied in these disorders and are crucial for the development
of cell and animal models used to study neurodegenera-
tion. Recently, the TAR DNA-binding protein 43 (TDP-43)
was identified as the disease accumulating protein in
patients with frontotemporal lobar degeneration with
ubiquitin inclusions (FTLD-U) and in amyotrophic lateral
sclerosis (ALS). TDP-43 was also found in the brains of 20–
30% of patients with Alzheimer’s disease (AD). Here, we
evaluated whether mutations in TDP-43 cause disease in a
cohort of 296 patients presenting with FTLD, ALS or AD. We
identified three missense mutations in three out of 92
familial ALS patients (3.3%), and no mutations in AD or
FTLD patients. All the identified mutations clustered in
exon 6, which codes for a highly conserved region in the C-
terminal part of the TDP-43 protein, which is known to be
involved in the interaction of TDP-43 with other proteins.
We conclude that mutations in TDP-43 are a rare cause of
familial ALS, but so far are not found in other neurode-
Table 1. Patients included in the TARDBP sequencing
Patients with positive
family history (N)
TDP-43 Mutations in ALS
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were further identified, none of which was predicted to affect the
TDP-43 protein (Table S1). Genomic TARDBP copy-number
analyses in 208 patients including all 116 ALS patients did not
reveal deletions or multiplications.
All TARDBP mutations identified in this study are located in
exon 6 (Figure 2). In the index patient of family A (ND10588), we
identified the known c.1009 A.G mutation, predicted to
substitute valine for methionine at codon 337 (p.M337V), and
previously reported to segregate with disease in a large British
autosomal dominant ALS kindred. In the index patient of family B
(ND08308), a novel mutation c.1035 C.A was identified,
predicted to change asparagine to a lysine at codon 345
(p.N345K). Finally, in the index patient of family C (ND08470),
a novel mutation c.1147 A.G which predicts an isoleucine for a
valine substitution at codon 383 (p.I383V) was identified.
Sequence analysis of TARDBP exon 6 in 185 healthy control
individuals did not identify these or other sequence variants. Using
custom made TaqMan genotyping assays, the presence of
p.M337V, p.N345K and p.I383V was further excluded in 640
US control individuals. Genotyping 652 sporadic ALS patients for
Figure 1. Missense mutations identified in TARDBP in familial ALS patients. (A) Pedigrees showing family history of ALS for three probands
carrying TARDBP mutations. Black symbols represent patients affected with ALS; white symbols represent unaffected individuals. Pedigrees are
constructed based on family history data provided by the NINDS Human Genetics Resource Center DNA and Cell Line Repository (http://ccr.coriell.org/
ninds). The alive/dead status of individuals is unknown. Arrowheads indicate the probands. The onset age of ALS symptoms and the TARDBP mutation
identified are included below each proband. (B) DNA sequence traces observed in a sample from the proband of each family. The observed single base
substitution and predicted amino acid change are indicated below each chromatogram. cDNA numbering is according to the largest TARDBP transcript
(NM_007375.3) and starting at the translation initiation codon. Protein numbering is relative to the largest TDP-43 isoform (NP_031401.1).
Figure 2. Overview of mutations identified to date in TARDBP. Schematic overview of the 7 TARDBP exons showing coding regions in dark
blue and non-coding regions in light blue (top). The TDP-43 protein structure with location of the conserved domains is shown with protein
numbering according to the largest isoforms NP_031401.1 (middle). Protein sequence alignment shows strong conservation in the C-terminal region
of TDP-43 (bottom). Colored boxes indicate the position of known and novel TDP-43 mutations identified in sporadic (orange) and familial (red) ALS
patients. TDP-43 mutations identified in this study are underlined. Orange and red lines in TARDBP gene and TDP-43 protein indicate approximate
positions of the mutations. RRM=RNA recognition motif.
TDP-43 Mutations in ALS
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these mutations did not identify additional mutation carriers. Since
all 3 mutation carriers were obtained from the National Institute of
Neurological Disorders and Stroke (NINDS) Human Genetics
Resource Center DNA and Cell Line Repository (Coriell), DNA
samples of relatives were unavailable for genetic studies and
segregation of the mutations with disease could therefore not be
Clinical Characteristics of TARDBP Mutation Carriers
All 3 TARDBP mutation carriers were identified in the clinical
patient series and were diagnosed by El Escorial criteria with
probable or probable-lab supported ALS. Electromyography
(EMG) examination was performed in 2 patients (ND10588 and
ND08470) and was supportive of the diagnosis of ALS. A detailed
overview of the distribution of upper and lower motor neuron
signs in the TARDBP mutation carriers is included in Table S2.
Patients ND10588 and ND08308 showed early onset ages of 38
and 39 years, respectively, while patient ND08470 showed
symptom onset at 59 years (Figure 1). The initial presenting
symptom in patients ND10588 and ND08470 was upper-limb
ALS, while ND08308 suffered from lower-limb onset ALS. No
signs of dementia or other atypical features of ALS were reported
in any of the mutation carriers or their affected relatives. No
autopsy of TARDBP mutation carriers was available.
Allele Sharing Analyses of TARDBP p.M337V
To investigate whether our US p.M337V mutation carrier and
the previously reported p.M337V family from the UK are
descendants of a common founder, we did an allele sharing study
with 12 short tandem repeat (STR) markers spanning a region of
6.7 Mb flanking TARDBP, including 5 markers within 1.0 Mb of
TARDBP (Table 2). We determined the disease haplotype in the UK
family and compared this to the genotypes observed in ND10588 to
detect allele sharing. Shared alleles were observed for 6 out of 12
STR markers in the region, however, only
(Chr1(AC)_11.06) directly flanking TARDBP was shared and the
(62.4%). In addition, potentially shared alleles at all other markers in
the region were alsocommon (.28%). These results makeit unlikely
that p.M337V originated from a common founder.
Biochemical Analysis of TARDBP Mutations in Familial ALS
Kabashi and colleagues previously reported a substantial
increase in a ,28 kDa fragment in lymphoblastoid cells with
TARDBP mutations in the presence of the proteasomal inhibitor,
MG-132, but not in lymphoblastoid cells derived from control
individuals or ALS patients suggesting an increase aggregation
property of these TDP-43 mutants . Based on this result, we
performed a similar study and analyzed the 3 patients with
TARDBP mutations identified in our study, 2 sporadic ALS cases
and 5 control individuals in the presence or absence of MG-132.
Consistent with the previous report, a marked increase in the
accumulation of detergent insoluble TDP-43 protein fragments
were observed in the lymphoblastoid cell lines treated with MG-
132 derived from patients with TARDBP mutations but not those
derived from control individuals. In our study, we sized the higher
and lower TDP-43 C-terminal fragments at approximately 35 and
25 kDa respectively (Figure 3). A similar increase was also found in
individuals with sporadic ALS (Figure 3).
43 by caspases can generate insoluble C-terminal fragments (35 and
25 kDa) similar to those found in diseased brains. Therefore, we
investigated whether proteasome-induced toxicity was associated
with proteolytic processing of endogenous TDP-43 in cell culture
models. H4 neuroglioma cells were treated with either vehicle
(DMSO) or proteasome inhibitor I (PSI) (10 mM) for 24 hours. In
the presence of PSI, TDP-43 was cleaved into ,35 and ,25 kDa
fragments (Figure 4), similar to the 35 and 25 kDa fragments found
in the lymphoblastoid cell lines derived from the TARDBP mutation
carriers (Figure 3). Similar results wereobtained using MG-132(data
not shown). The inhibitory activity and toxicity of PSI also led to a
marked increase in cleaved (active) capase-3 levels, which promotes
apoptotic cell death and accumulates upon such inhibition.
Furthermore, when we co-treated the cells with PSI and the caspase
inhibitor, Z-VAD (OMe)-FMK, the generation of proteolytic TDP-
43 fragments was inhibited (Figure 4). HSP70 immunoblot analysis
was used to verify the inhibition of the proteasomal machinery. As
expected, HSP70 levels were increased after PSI treatment and the
levels persisted in the presence of caspase inhibitor Z-VAD (OMe)-
FMK (Figure 4). Taken together, these data strongly suggest that
proteasome inhibition is sufficient to promote proteolytic cleavage
and accumulation of TDP-43 through a mechanism that implicates
programmed cell death.
The identification of rare mutations in genes encoding the
major protein component of the pathologic brain depositions
observed in familial neurodegenerative diseases has played a
critical role in our current understanding of the molecular
pathways underlying AD (APP), FTLD (MAPT) and Parkinson’s
disease (SNCA) [33,34]. In this study, we performed mutation
analyses of TARDBP, encoding TDP-43, in a large cohort of
patients with neurodegenerative diseases characterized by TDP-43
pathology to determine if rare mutations or multiplications in
TARDBP are involved in the genetic etiology of TDP-43
proteinopathies. Patients with a clinical diagnosis of ALS, FTLD
or FTLD-ALS, and patients with pathologically confirmed TDP-
43-proteinopathy were included in the analyses. In support of our
Table 2. Allele sharing in p.M337V families.
D1S26637.18 2018.9 189–199
D1S2694 7.26 23850.0238–238
D1S1635 10.91 16014.8 147–157
c.1009A.G (p.M337V) 11.01G-A–G
Chr1_11.06 11.06 26462.4264–270
Chr1_11.28 11.28 12814.0 132–132
D1S266711.41 26428.6 260–264
D1S2740 11.8490 57.490–100
D1S489 11.9714337.5 143–143
D1S434 12.25240 1.8246–248
D1S22813.86121 26.8 119–123
aGenomic position relative to the UCSC genome browser on the Human Mar.
2006 Assembly (http://genome.ucsc.edu/).
bAlleles that are shared between the UK family and patient ND10588 are in
TDP-43 Mutations in ALS
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hypothesis, 14 different pathogenic TARDBP missense mutations
were reported by other researchers during the course of this study
in familial and sporadic ALS patients [30–32,35].
We identified 2 novel TARDBP missense mutations (p.N345K and
p.I383V) and the known p.M337V mutation in 3 out of 92 familial
ALS patients (3.3%), while no mutations were identified in 24
sporadic ALS patients or 180 patients with other neurodegenerative
diseases. p.M337V, p.N345K and p.I383V were excluded in 825 US
control individuals and in 652 additional sporadic ALS patients. The
TARDBP mutation frequency in our familial ALS cohort is
comparable to the frequency reported by Kabashi and colleagues
 (3/80 patients=3.8%) but considerably higher than the
frequency reported by Sreedharan and colleagues (1/154 pa-
tients=0.6%) . This may reflect the difference in study design,
as a significant number of our patients were index patients of
autosomal dominant ALS families, including all 3 patients carrying
TARDBP mutations. Unfortunately, since all mutation carriers were
index patients obtained from the NINDS Human Genetics Resource
not available to determine segregation of the mutations with disease.
The absence of TARDBP mutations in patients with neurodegener-
ative diseases other than ALS in our study, confirms the lack of
mutations and genetic association of TARDBP in FTLD populations
[30,36–38]. However, without extensive TARDBP sequence analyses
in additional cohorts of FTLD and AD patients, TARDBP mutations
cannot be excluded as a rare cause of these disorders.
All TARDBP mutation carriers identified in this study presented
with probable ALS according to El Escorial criteria in the absence
Figure 3. Biochemical analysis of TDP-43 in lymphoblastoid cell lines of TARDBP mutation carriers. Western blot analyses of protein
lysates derived from lymphoblastoid cell lines from 3 familial ALS patients carrying different TARDBP mutations (p.M337V, p.N345K and p.I383V), 2
ALS patients (1 and 2) without TARDBP mutations and 5 healthy control individuals (Control 1–5). In lymphoblastoid cell lines derived from TARDBP
mutation carriers and sporadic ALS patients an accumulation of 2 smaller C-terminal fragments of TDP-43 protein of approximately 35 and 25 kDa
was observed in detergent-insoluble fractions treated with the proteasome inhibitor, MG-132. In lymphoblastoid cell lines derived from control
individuals the levels of the 35 kDa fragment were substantially lower, and the 25 kDa fragment was mostly undetectable. Membranes from the
soluble fraction were reprobed for beta-actin to monitor protein loading.
Figure 4. Proteasome inhibition increases the proteolytic
cleavage of TDP-43. Western blot analyses of H4 neuroglioma cells
treated with the proteasome inhibitor, PSI (10 mM, 24 hours) and a pan-
caspase inhibitor, Z-VAD-FMK (100 mM, 24 hours) separately or in
combination. Treatment with PSI revealed an increase in proteolytic
cleavage of TDP-43 fragments (35 and 25 kDa) and an increase in
caspase-3 activity. Treatment with a pan-caspase inhibitor suppressed
PSI-induced TDP-43 cleavage and caspase-3 activity. HSP70 levels were
increased after PSI treatment and the levels persisted in the presence of
a pan-caspase inhibitor. Similar results were obtained in 3 independent
TDP-43 Mutations in ALS
PLoS Genetics | www.plosgenetics.org5 September 2008 | Volume 4 | Issue 9 | e1000193
of atypical clinical signs, in agreement with the previous reports on
TARDBP mutation carriers.
Thep.M337V mutation haspreviouslybeenreported tosegregate
with disease in a British autosomal dominant ALS family . We
identified p.M337V in an index patient from a US family with a
strong family history of ALS. Our mutation carrier showed upper
limb-onset ALS at 38 years of age, 6 years younger than the earliest
onset age reported in the British p.M337V family. Signs of dementia
were not reported in any of the family members, consistent with the
previous report. An allele sharing study using 12 STR markers
flanking TARDBP did not support a common ancestor for the UK
family and our US patient, although our set of analyzed markers
would not have detected a very distant common ancestor [39,40]. In
addition, we cannot exclude the rare possibility that marker
Chr1_11.28 mutated in patient ND10588 or that the genomic
position of this marker is incorrect, which would leave open the
possibility of a shared region of maximum 1.3 Mb (D1S1635-
D1S434). In anyway, the identification of p.M337V in two
genealogically unrelated ALS families adds further strength to the
pathogenicity of TARDBP mutations and justifies mutation screening
for TARDBP in patients with familial ALS.
Similar to 13 of the 14 previously reported TARDBP mutations,
both novel missense mutations identified in this study were located
in exon 6 encoding the highly conserved C-terminus of TDP-43,
known to be involved in protein-protein interactions (Figure 2).
p.N345K was identified in a 43 year old male with a 4 year history
of ALS and an autosomal dominant family history. The p.I383V
mutation was also identified in a familial ALS patient; however the
onset age was 59 years, 2 decades later than the other 2 mutations
identified in this study. This may reflect the more conservative
amino acid substitution (IsoRVal) or its more C-terminal location
in the TDP-43 protein compared to the other mutations, which
may induce a different disease mechanism. Alternatively, addi-
tional genetic and/or environmental factors may determine the
disease expression of TARDBP mutations, as suggested by the wide
onset age range (48–83 years) observed in the recently published
family with the p.A315T mutation in TARDBP . Finally,
although there is strong evidence supporting that p.N345K and
p.I383V are pathogenic, there remains the possibility that these
mutations in fact represent rare benign polymorphisms. Definitive
confirmation of their pathogenic nature will depend on finding
additional ALS patients carrying these mutations.
To determine the pathological significance of TARDBP missense
mutations on the post-translational processing of TDP-43, we
examined human lymphoblastoid cell lines derived from all 3 familial
TARDBP mutation carriers identified in this study, 2 ALS patients
without TARDBP mutations and 5 control individuals (Figure 3).
Patient cell lines revealed a substantial increase in a proteolytic
cleaved fragment with a molecular weight of approximately 35 and
25 kDa consistent with caspase cleavage . These data suggest that
TARDBP mutations may cause a toxic gain of function through novel
protein interactions or intracellular accumulation, particularly of
caspase fragments. Kabashi and colleagues previously reported a
similar substantial increase in a fragment of approximately 28 kDa in
lymphoblastoid cell lines of TARDBP mutation carriers. This
fragment accumulated in the presence of a proteasome inhibitor
(MG-132), which led the authors to speculate that this TDP-43
product is likely degraded by the ubiquitin-proteasome system (UPS)
. While we can’t exclude the enhanced aggregation of their
mutants in the presence of the inhibitor, our data suggests that
proteasome-induced toxicity enhances proteolytic cleavage of TDP-
43 into 35 and 25 kDa fragments, resulting in cleavage fragments
similar to those observed in ALS patients (Figure 4). Although we
can’t exclude the possibility that these fragments may be degraded by
the UPS, it is likely that the accumulation of these fragments is
primarily mediated by caspase cleavage.
In conclusion, our findings support that TARDBP mutations are
a rare cause of ALS, but so far are not found in other
neurodegenerative diseases. Since all reported TARDBP mutations
cluster in exon 6 encoding a highly conserved region of the TDP-
43 protein, selective mutation analyses of TARDBP exon 6 in
familial and sporadic ALS may be warranted.
Materials and Methods
Our initial study population comprised a total of 296 patients with
TDP-43 related neurodegenerative diseases, including 176 clinically
diagnosed patients with ALS, FTLD and FTLD-ALS and 120
patients with pathologically confirmed TDP-43 proteinopathy. The
average age at onset in the clinical cohort was 57.8610.7 (range 31–
81 years)and the average age at death inthe pathological cohort was
74.8613.8 (range 38–100 years). Among patients with known
ethnicities (N=214), 95% were Caucasian (N=203), 3% were
Hispanic (N=7) and 2% were others (African/American (N=2),
East-Indian (N=1) and Caribbean (N=1)). A summary of the
primary diagnoses and family history of the patients is provided in
Table 1. The majority of the pathological confirmed patients
(N=87) were derived from the Mayo Clinic Jacksonville Brain Bank
and primarily ascertained through The State of Florida Alzheimer’s
Disease Initiative funded through the Department of Elder Affairs,
The Einstein Aging Study, The Udall Center for Excellence in
Supranuclear Palsy, the Mayo Alzheimer’s Disease Patient Registry
(ADPR) and the Florida Alzheimer’s Disease Research Center
(ADRC). Additional clinical and pathological confirmed patients
were ascertained through the Mayo Clinic Jacksonville and
Rochester ADRC (N=60), Mayo Clinic Scottsdale Alzheimer’s
Disease Center (ADC) (N=4), the Neurological Institute of New
York, Columbia University (N=2), the University of California, Los
Angeles (UCLA) ADC (N=23), the University of British Columbia
(N=58), the Harvard Brain Bank (N=5), the Sun Health Research
Institute (N=4), the Drexel University College of Medicine (N=1),
the Northwestern Feinberg School of Medicine (N=13) and the
Coriell Institute for Medical Research (N=39). A list of the specific
samples from the Coriell Institute included in the TARDBP mutation
screening is provided as Table S3.
To determine the frequency of the TARDBP mutations
identified in our initial cohort, an additional cohort of 652
sporadic ALS patients was obtained from the University of British
Columbia (N=140), the Neurological Institute of New York,
Columbia University (N=48) and the Coriell Institute for Medical
Research (N=464). All control individuals (N=825) included in
the study were Caucasian and ascertained through the Mayo
Clinics in Jacksonville, Florida and Scottsdale, Arizona.
The 5 coding and 2 non-coding exons of TARDBP were amplified
by polymerase chain reaction (PCR) in standard 25 ml reactions
using Qiagen PCR products (Table S4). PCR products were purified
using the Agencourt Ampure method and sequenced using Big dye
terminator V.3.1 products. Sequencing products were purified using
the Agencourt CleanSEQ method and analyzed on an ABI 3730
DNA analyzer (Applied Biosystems, Foster City, CA, USA).
The presence of TARDBP mutations c.1009A.G (p.M337V),
c.1035 C.A (p.N345K) and c.1147 A.G (p.I383V) in sporadic
TDP-43 Mutations in ALS
PLoS Genetics | www.plosgenetics.org6 September 2008 | Volume 4 | Issue 9 | e1000193
ALS patients and control individuals was determined with custom-
designed TaqMan SNP genotyping assays (Applied Biosystems)
(Table S5) and analyzed on an ABI7900 genetic analyzer using
TARDBP Copy-Number Analyses
TaqMan gene expression assays to exons 2, 4 and 6 of TARDBP
and to exon 5 of PSEN2 (for use as endogenous control) were
designed using File Builder 3.1 software (Applied Biosystems) (Table
S6) to test for the presence of genomic TARDBP copy-number
mutations in 208 patients selected from our population. This
approach was used to detect copy-number mutations affecting exons
2, 4 or 6, as well as complete TARDBP and large N- and C-terminal
TARDBP deletions and multiplications. Real-time PCR with 25 ng
genomic DNA as template was performed on an ABI7900 using the
TaqMan method according to standard procedures. All samples
were run in triplicate. The FAM-fluorescent signal was analyzed
using SDS2.2.2 software, and genomic copy number determined by
relative quantification (DDct method).
p.M337V Allele Sharing Studies
To examine whether the US and UK families carrying the
p.M337V mutation shared a common founder, we typed 12 STR
markers spanning a region of 6.7 Mb flanking TARDBP in3 patients
and 8 unaffected relatives of the previously published UK family, in
the US patient ND10588 and in 2 CEPH samples. STR markers
were amplified with one fluorescently labeled primer and PCR
fragments were analyzed on an automated ABI3100 DNA analyzer.
Alleles were scored using the Genemapper software (Applied
Biosystems). CEPH allele frequencies were used to estimate the
allele frequency of the shared alleles in control individuals (CEPH
genotype database; http://www.cephb.fr/cephdb/). The 2 novel
markers were PCR amplified using Chr1_11.06-F: FAM-CAG-
CATCATGTGGTTTGGCAGT, Chr1_11.06-R: CAGCTCG-
CAGGGAAGATGAAA, Chr1_11.28-F: FAM-TGGCCATCT-
TAACAGGAACAGC and Chr1_11.28-R:TTCAAGGGCTTTC-
GAGGTGAA and allele frequencies were estimated in a population
of 93 unrelated US control individuals.
Cell Culture and Treatment
H4 neuroglioma cells were grown in Opti-Mem plus 10% FBS
and 1% pen-strep. Cells were plated in 6-well plates and at 90%
confluency treated with 10 mM proteasome inhibitor I (PSI) (EMD
Chemicals, Inc. San Diego, CA) or 100 mM pan-caspase inhibitor
(Z-VAD-FMK) (EMD Chemicals, Inc. San Diego, CA) separately
or in combination. Twenty-four hours after treatment, cells were
harvested for subsequent Western blot analysis in the Co-IP buffer
(50 mM Tris-HCl, pH 7.4, 1 M NaCl, 1% Triton-X-100, 5 mM
EDTA) plus 1% SDS, PMSF, protease and phosphatase inhibitors.
A similar experiment was performed using 10 mM MG-132
(Calbiochem, San Diego, CA) instead of PSI.
Lymphoblastoid cells from 5 healthy control individuals, 3
familial ALS patients with TARDBP mutations and 2 ALS patients
without TARDBP mutations were grown in RPMI1640 plus 10%
FBS and 1% pen-strep. Cells were plated in T25 flasks and treated
the following day with MG-132 (20 mM, 6 hours). Cell pellets
from each cell line were lysed with the 0.2% Triton X-100-PBS
with PMSF, protease and phosphatase inhibitors on ice for
10 minutes. After sonication, samples were centrifuged at 10,000 g
for 15 minutes at 4uC. The supernatant was saved as the soluble
fraction and the pellet was resuspended, sonicated in 2% SDS-
PBS-Urea and saved as the insoluble fraction. The soluble and
insoluble fractions were subjected to Western blot analysis.
Western Blot Analysis
Protein concentrations of cells lysates were measured by a
standard BCA assay (Pierce, Rockford, IL). Next, samples were
heated in Laemmli’s buffer and equal amounts of protein were
loaded into 10-well 10% or 4–20% Tris-glycine gels (Novex, San
Diego, CA). After transfer, blots were blocked with 5% nonfat dry
milk in TBST (TPS plus 0.1% Triton X-100) for 1 hour, and then
incubated with rabbit polyclonal TDP-43 antibody (1:500;
ProteinTech Group, Inc, Chicago, IL), rabbit polyclonal cas-
pase-3 antibody (1:1000; Cell Signaling, Beverly, MA), HSP70
(1:2000; Stressgen, Ann Arbor, MI) or mouse monoclonal b-actin
antibody (1:5000, Sigma, Saint Louis, MS) overnight at 4uC.
Membranes were washed three times each for 10 minutes with
TBST and then incubated with anti-mouse or anti-rabbit IgG
conjugated to horseradish peroxidase (1:2000; Jackson ImmunoR-
esearch, West Grove, PA) for 1 hour. Membranes were then
washed three times each for 10 minutes, and protein expression
was visualized by ECL treatment and exposure to film.
Found at: doi:10.1371/journal.pgen.1000193.s001 (0.07 MB
Sequence variants identified in TARDBP.
in TARDBP mutation carriers.
Found at: doi:10.1371/journal.pgen.1000193.s002 (0.03 MB
Distribution of Upper and Lower Motor Neuron signs
the TARDBP mutation analyses.
Found at: doi:10.1371/journal.pgen.1000193.s003 (0.07 MB
Specific samples from the Coriell Institute included in
Found at: doi:10.1371/journal.pgen.1000193.s004 (0.03 MB
TARDBP PCR and sequencing primers.
Found at: doi:10.1371/journal.pgen.1000193.s005 (0.03 MB
Primers and probes for TARDBP copy-number
Found at: doi:10.1371/journal.pgen.1000193.s006 (0.03 MB
Detailed Information on TARDBP Taqman genotyp-
We would like to thank the families who contributed samples that were
critically important for this study. ALS samples from the National Institute
of Neurological Disorders and Stroke (NINDS) Human Genetics Resource
Center DNA and Cell Line Repository (http://ccr.coriell.org/ninds) and
the ALS Research Group (ALSRG) were included in this study.
Conceived and designed the experiments: NJR MB MH LP RR.
Performed the experiments: NJR YJZ MB JMG NAF YFX RJC LP RR.
Analyzed the data: NJR IRM LP RR. Contributed reagents/materials/
analysis tools: HS BJK KK JS CV ES CL EHB DHG DSK HM RCP
NRC CES KBB BB NRGR ZKW RJC DWD LP RR. Wrote the paper:
NJR YJZ RJC KBB NRGR ZKW DWD IRM LP RR.
TDP-43 Mutations in ALS
PLoS Genetics | www.plosgenetics.org7September 2008 | Volume 4 | Issue 9 | e1000193
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