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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Neurobiol Aging. Author manuscript; available in PMC 2012 November 1.
Published in final edited form as:
PMCID: PMC2889148

Mutational Analysis of TARDBP in Neurodegenerative Diseases


Neurodegenerative diseases are often characterized by the presence of aggregates of misfolded proteins. TDP-43 is a major component of these aggregates in Amyotrophic Lateral Sclerosis (ALS), but has also been observed in Alzheimer's (AD) and Parkinson's Diseases (PD). In addition, mutations in the TARDBP gene, encoding TDP-43, have been found to be a significant cause of familial ALS (FALS). All mutations, except for one, have been found in exon 6. To confirm this observation in ALS and to investigate whether TARDBP may play a role in the pathogenesis of AD and PD, we screened for mutations in exon 6 of the TARDBP gene in three cohorts composed of 376 AD, 463 PD (18% familial PD) and 376 ALS patients (50% FALS). We found mutations in ~7% of FALS and ~0.5% of sporadic ALS (SALS) patients, including two novel mutations, p.N352T and p.G384R. In contrast, we did not find TARDBP mutations in our cohort of AD and PD patients. These results suggest that mutations in TARDBP are not a significant cause of AD and PD.

1. Introduction

Neurodegenerative diseases are often characterized by the presence of intracellular or extracellular protein aggregates in the central nervous system. An evolving molecular classification of these disorders is based upon the biochemical nature of the proteins forming the inclusions. The presence of tau-negative, ubiquitinated inclusions (UBIs) in the perikaryon and proximal axons of surviving motor neurons is the neuropathological hallmark of amyotrophic lateral sclerosis (ALS) and indicates a failure of the proteasome to recycle damaged proteins (Leigh, et al., 1991). UBIs are also observed in cortical neurons of patients with tau-negative and ubiquitin-positive frontotemporal lobar dementia (FTLD-U), the most common neuropathological presentation of FTLD (Forman, et al., 2006).

TDP-43 is a protein of 43 kDa (414 residues) encoded by the TARDBP gene that has been identified as the major component of UBIs in brain tissues from ALS and FTLD-U patients (Neumann, et al., 2006). Its involvement in both disorders is consistent with the view that these two neurodegenerative conditions share common molecular pathways. TDP-43 belongs to the family of heterogeneous nuclear ribonucleoproteins (hnRNPs) and has been identified as a transcriptional repressor (Ou, et al., 1995) and as promoting exon skipping (Buratti, et al., 2001). Interestingly, mutations in the TARDBP gene have been described as major cause of familial ALS (FALS) and have also been identified in sporadic ALS cases (SALS) and FTLD with associated motor neuron disease (Benajiba, et al., 2009, Kabashi, et al., 2008, Sreedharan, et al., 2008, Van Deerlin, et al., 2008). With only one exception, the mutations have been found in exon 6.

By contrast with TARDBP gene mutations, which have been described only in ALS and FTLD-U, TDP-43 positive inclusions have been observed in diverse neurodegenerative disorders including not only FTD and ALS, but also Alzheimer's disease (AD), alpha-synucleinopathies (such as Parkinson's disease and dementia with Lewy bodies), and tauopathies (corticobasal degeneration). In autopsy studies, it is estimated that as many as 20% of AD (Amador-Ortiz, et al., 2007) and ~7% of Parkinson's disease (PD) (Nakashima-Yasuda, et al., 2007) cases reveal evidence of TDP-43 immunoreactivity in the central nervous system. These findings raise the hypothesis that primary germline TARDBP mutations may be present in neurodegenerative diseases other than ALS and FTLD-U. Although no TARDBP mutations have been identified so far in those populations (Kabashi, et al., 2009,Rutherford, et al., 2008,Van Deerlin, et al., 2008), it is possible that the cohorts studied were underpowered. To investigate this hypothesis, we screened the TARDBP gene for exon 6 mutations in two cohorts of AD and PD patients. To better assess the role of TARDBP in the pathogenesis of these disorders, we also screened a set of FALS and SALS patients.

2. Materials and methods

2.1 Study population

To determine whether TARDBP mutations are present in AD and PD, we obtained two cohorts of 376 AD patients and 463 PD patients, with IRB approval. All AD samples were diagnosed with definite AD according to the NINCDS-ADRDA criteria (McKhann, et al., 1984) and were obtained from the Massachusetts General Hospital Alzheimer Disease Research Center Tissue Repository. No information is available regarding the family history of the AD cases screened. PD samples were collected at the Coriell Institute for Biomedical Research Cell Repositories (panels NDPT005, NDPT015 and NDPT016) and at the Massachusetts General Hospital Institute for Neurodegenerative Diseases. The PD cohort included 82 patients (18%) with a positive family history for Parkinsonism. All the patients met the UK Brain Bank Criteria for idiopathic PD (Hughes, et al., 1992), and clinical records were reviewed by a neurologist with expertise in movement disorders. Additionally, we screened DNA from 397 ALS patients (208 FALS and 188 SALS) who had been diagnosed with ALS, according to the revised El Escorial criteria (Miller, et al., 1999). All FALS patients tested negative for mutations in the SOD1 and FUS/TLS genes.

2.2 Molecular analysis

Mutational screening of exon 6 of TARDBP was performed by touchdown PCR using primers TDP43E×6F (agtaaaacgacggccagttgaatcagtggtttaatcttctttg) and TDP43E×6R (gcaggaaacagctatgaccaaaatttgaattcccaccattc). These primers anneal to adjacent intronic and 3′UTR regions of exon 6 and contain 5′ tails encoding M13 forward and reverse. PCR-products were subsequently purified by incubation with Exonuclease I and Shrimp Alkaline Phosphatase, sequenced with M13 primers using the BigDyeTerminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA USA) and then resolved by capillary electrophoresis on an ABI 3730XL DNA Analyzer (Applied Biosystem). Sequence analysis was performed using the PHRED/PHRAP/Consed software suite ( and variations in the sequences were identified with the Polyphred v6.15 software (

3. Results

In our ALS cohort, the screening of exon 6 of the TARDBP gene revealed 10 different heterozygous missense mutations in 14 patients (Table 1 and Supplemental Table S1). Three of the mutations observed were novel (N352T, N378D and G384R), although the substitution of asparagine 352 with a serine has been previously described in a German kindred (Kuhnlein, et al., 2008). In contrast, the screening of both the PD and AD cohorts did not reveal the any mutations within the coding region of exon 6.

Table 1
TARDBP mutations within Familial and Sporadic ALS.

With the exception of G295R, which has been identified in a single SALS individual, all other mutations have been found exclusively in FALS cases. Thus, the frequency of TARDBP mutations in our cohort is more than ten-times higher in FALS than SALS. Our observation that TARDBP mutations account for ~6.3% of all SOD1 and FUS/TLS negative FALS patients is consistent with the results of previous studies (Corrado, et al., 2009). On the contrary, we observed a lower-than-expected TARDBP mutational frequency in SALS (~0.5%) in comparison with other screenings that reported mutations in ~2% of sporadic cases (Corrado, et al., 2009, Daoud, et al., 2009, Van Deerlin, et al., 2008).

Three variants (I383V, G348C, and N352T) account for half of the TARDBP mutations identified in our cohort and are present in ~1% of FALS patients respectively. The majority of the 30 TARDBP mutations described so far have been identified in isolated kindred or sporadic cases. The most frequently observed mutation, A382T, has been reported in nine patients of French and Italian origin (Corrado, et al., 2009, Kabashi, et al., 2008). We did not detect A382T in our cohort, possibly due to a different ethnic background of our patients.

With the exception of I383V, all the detected mutations change residues that are conserved throughout evolution. Accordingly, they are predicted to have a deleterious effect on protein structure or function by at least one of the two following in silico software applications: SNAP ( and PMut ( Although not fully conserved, we hypothesize that the substitution of isoleucine 383 with valine is pathogenic, since the mutation has been observed in four FALS cases (Rutherford, et al., 2008), including ours, but not in 4252 healthy controls from different populations (Corrado, et al., 2009, Kabashi, et al., 2008, Kuhnlein, et al., 2008, Sreedharan, et al., 2008, Van Deerlin, et al., 2008).

4. Discussion

Our mutational screening failed to identify TARDBP exon 6 mutations within a cohort of 376 AD and 463 PD patients. However, within ALS samples we identified seven previously described TARDBP mutations and three novel mutations, N352T, N378D and G384R, all affecting residues conserved throughout evolution and predicted to be pathogenic by in silico analyisis. The TARDBP mutations represent ~6.3% of our FALS cohort. Since SOD1 and FUS/TLS mutations are observed in ~25% of ALS pedigrees, we estimate a mutational frequency for TARDBP of 4.7%. These findings are consistent with previous reports (Corrado, et al., 2009), and substantiate the observation that TARDBP mutations represent the second most frequent cause of FALS after SOD1. In the SALS cohort, we observed a G295R mutation in a single patient, for a mutational frequency of ~0.5%. Our observations thus differ from other studies that report TARDBP mutations in ~2% of all SALS cases (Corrado, et al., 2009, Daoud, et al., 2009, Van Deerlin, et al., 2008). The discrepancy may reflect the differing ethnic backgrounds among the patient cohorts studied to date. Alternatively, it may reflect a statistically insignificant variant; because TARDBP variants are much less frequent in SALS than FALS, larger cohorts are likely required to correctly assess their mutational frequency in SALS.

Although TDP-43 positive inclusions have been described in neurons of patients affected with AD, PD and other neurodegenerative diseases, TARDBP mutations have been observed only in ALS with or without associated FTLD. Three previous reports could not find mutations in 46 AD (Rutherford, et al., 2008), 125 PD (Kabashi, et al., 2009), and 41 patients with neurodegenerative diseases other than ALS or FTLD-U (Van Deerlin, et al., 2008). Given the low mutational frequency of TARDBP in sporadic cases, we attempted to validate these results in two larger AD and PD cohorts. Given that we also failed to find exon 6 mutations in our cohort, it seems unlikely that TARDBP mutations in exon 6 are involved in the pathogenesis of AD and PD. Our rationale in screening only exon 6 of the TARDBP gene relies on the observation that almost all the ALS-associated mutations are clustered in the C-terminal region of the gene. Moreover, in neuronal inclusions TDP-43 is abnormally cleaved to generate 25kDA C-terminal fragments (Neumann, et al., 2006), suggesting that the region encoded by exon 6 is crucial for the formation of aggregates. We cannot, however, exclude that mutations in other regions of the gene may be associated to AD and PD pathogenesis. Also, since the frequency of TDP-43 inclusions in idiopathic PD is ~7%, it is possible that our PD cohort, while considerably larger than those previously studied, is still underpowered to detect TARDBP mutations. Further studies are necessary to determine if TDP-43 is an innocent bystander that is co-precipitated in aggregates or, more interestingly, if the protein is actively involved in the pathogenic pathways leading to AD and PD. Supporting the latter hypothesis is the evidence that the presence of a TDP-43 abnormal immunoreactivity is associated with a modified AD phenotype characterized by a greater cognitive impairment and a more pronounced hippocampal atrophy (Josephs, et al., 2008).

Supplementary Material


A special thanks to the patients and their caregivers. Generous support was provided 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 NINDS (NS050557). NT and VS were supported by the Italian Ministry of Health (Malattie Neurodegenerative, ex Art.56, n.533F/N1). PS was supported by the Howard Hughes Medical Institute (HHMI) through the auspices of Prof. H. Robert Horvitz, an Investigator in the HHMI. The MGH PD DNA Bank is supported by the National Parkinson Foundation and the NIH (5P50NS038372-10).


Disclosure Statement: RHB is a co-founder of AviTx, which targets development of therapies.

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