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
. 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 hypothesis, 14 different pathogenic TARDBP
missense mutations were reported by other researchers during the course of this study in familial and sporadic ALS patients 
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 
3.8%) but considerably higher than the frequency reported by Sreedharan and colleagues (1/154 patients
. 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 Center DNA and Cell Line Repository, DNA of affected relatives was not available to determine segregation of the mutations with disease. The absence of TARDBP
mutations in patients with neurodegenerative diseases other than ALS in our study, confirms the lack of mutations and genetic association of TARDBP
in FTLD populations 
. 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 of atypical clinical signs, in agreement with the previous reports on TARDBP mutation carriers.
The p.M337V mutation has previously been reported to segregate 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 
. 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 (). 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 (Iso→Val) or its more C-terminal location in the TDP-43 protein compared to the other mutations, which may induce a different disease mechanism. Alternatively, additional 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 (). 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 (). 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.