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To identify novel causes of familial neurodegenerative diseases, we extended our previous studies of TAR DNA-binding protein 43 (TDP-43) proteinopathies to investigate TDP-43 as a candidate gene in familial cases of motor neuron disease. Sequencing of the TDP-43 gene led to the identification of a novel missense mutation, Ala-315-Thr, which segregates with all affected members of an autosomal dominant motor neuron disease family. The mutation was not found in 1,505 healthy control subjects. The discovery of a missense mutation in TDP-43 in a family with dominantly inherited motor neuron disease provides evidence of a direct link between altered TDP-43 function and neurodegeneration.
Motor neuron disease (MND) is a neurodegenerative disorder involving the loss of upper and/or lower motor neurons, and it is characterized clinically by progressive weakness and death within a few years of onset; the most common clinical MND phenotype is amyotrophic lateral sclerosis (ALS). Recently, TAR DNA-binding protein 43 (TDP-43) was identified as the major pathological protein of the motor neuron inclusions found in sporadic MND and also in frontotemporal lobar degeneration with ubiquitin-immunoreactive, tau-negative inclusions (FTLD-U), which can be associated with MND, but not in familial MND with Cu/Zn superoxide dismutase-1 (SOD1) mutation.1-4
Although largely sporadic, about 10% of MND cases are familial, and of these about 20% have mutations in the SOD1 gene.5 Evidence suggests that SOD1 mutations cause MND by a toxic gain of function.6 The recent discovery that pathological TDP-43 inclusions are present in sporadic/non-SOD1 cases of MND, but absent from SOD1 cases and SOD1 transgenic mice, suggests that the sporadic form of the disease may have a different underlying pathophysiology. Therefore, new genetic insights into MND are needed to further the understanding of disease pathogenesis and to develop animal models representative of the sporadic form of the disease.
Familial forms of neurodegenerative diseases can carry pathogenic mutations in the genes encoding the proteins present in the inclusions characterizing the disorder, for example, amyloid precursor protein in Alzheimer's disease,7 α-synuclein in Parkinson's disease,8 and tau in FTLD with tauopathy.9 Given that TDP-43-positive inclusions are present in most cases of sporadic and familial ALS, FTLD-MND, and FTLD-U, this gene is a strong biological candidate gene for familial forms of these disorders.
TDP-43 protein structure is evolutionarily conserved and consists of two RNA recognition motifs and a glycine-rich domain10 (Fig, A). TDP-43 can bind DNA and RNA, is involved in exon skipping of cystic fibrosis transmembrane conductance regulator (CFTR) gene, and binds to human immunodeficiency virus type 1 TAR DNA sequence motifs.11-13
In families of European descent, we have undertaken mutation analysis of the TDP-43 gene in 8 families with MND/ALS with an autosomal dominant pattern of inheritance and no mutation within the SOD1 gene, 5 families with familial FTLD-MND, and 25 families with FTLD-U.14 No sporadic cases of MND, FTLD-MND, or FTLD-U were screened. In brief, high-molecular-weight DNA was extracted from whole blood, serum, or brain tissue according to standard procedures. DNA from serum was whole-genome amplified using the REPLI-g Midi Kit (Qiagen, Valencia, CA) before genetic analysis. DNA from a single affected individual from each family was used for sequencing of TDP-43. All the exons and the intronexon boundaries of TDP-43 gene were amplified using gene-specific intronic primers. Direct sequencing of the amplified fragments was performed using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Wellesley, MA) and standard protocols. For most of the fragments, the primers used for sequencing were the same as those used for polymerase chain reaction amplification (primer sequences available on request). Reactions were run on an ABI3130, and mutation analysis was performed using Sequencher software v4.6 (Gene Codes Corporation, Ann Arbor, MI). Positive calls for sequence variants were made only if the variant was observed in both forward and reverse sequence reads. Where possible, sequence variants were tested for segregation with the disease and screened in a set of 1,505 unrelated ethnically matched control subjects.
This analysis led to the identification of a novel missense mutation, Ala-315-Thr (c.1077 G>A), within exon 6. In TDP-43, this alanine residue is highly conserved through the evolutionary spectrum from Homo sapiens to Xenopus tropicalis, supporting its likely functional importance (see Fig, B). The A315T mutation segregated with all affected members of an autosomal dominant MND family (additional noncoding sequence variants were also identified in cases with FTLD-U, FTLD-MND, and MND; see Supplementary Table 1 and Figs, C, D). This mutation was absent from a large series of ethnically matched elderly control subjects (n = 1,505).
The phenotype of the four affected family members with TDP-43 A315T mutation involved a slowly progressive lower motor neuron degeneration syndrome with respiratory involvement, with only minimal involvement of upper motor or bulbar neurons and absence of dementia (Table). Brain autopsy in this kindred remains to be undertaken. Similar clinical phenotypes have been reported in sporadic MND and in kindreds with SOD1 mutations.5,6
The TDP-43 mutation in familial MND reported here supplements other familial neurodegenerative conditions that affect predominantly lower motor neurons including mutations in the vesicle-associated membrane protein-associated protein B (VAPB), dynactin (DCTN1), alsin (ALS2), immunoglobulin μ-binding protein 2 (IGHMBP2), and glycyl-tRNA synthetase (GARS) genes, and other mutations in juvenile MND, although some of these mutations have been identified in MNDs and hereditary motor neuropathies with variable clinical phenotypes.6
These data have important implications for both sporadic and familial forms of MND and FTLD-U, which are linked by a common molecular pathology: TDP-43 proteinopathy. The discovery of a missense mutation in TDP-43 in a family with dominantly inherited MND provides evidence of a direct link between TDP-43 function and neurodegeneration, and should facilitate the generation of in vitro and transgenic models to enable the further elucidation of mechanisms in the pathogenesis of the TDP-43 proteinopathies. In addition, they may generate novel targets for therapeutic intervention.
This work was supported by the National Institutes of Health (P50 AG05681 (J.C.M., N.J.C.), P01 AG03991, (J.C.M., A.M.G., N.J.C.), U01 AG16976 (E.H.B., K.J.H., C.L.W., N.J.C.), P30 AG13854 (E.H.B.), P30 NS057105 (A.M.G., N.J.C.), K12 RR023249 (R.H.B.), P50 AG16574 (R.R), and R01 MH57899-01A1 (R.C.)), the Arizona Alzheimer's Disease Research Consortium (R.C.), the Hope Center for Neurological Disorders (A.M.G., N.J.C.), the Buchanan Fund (N.J.C.), and the Barnes-Jewish Hospital Foundation (N.J.C.).
We thank the clinical, genetic, pathology, and technical staff of the collaborating centers for making information and DNA/tissue samples available for this study. We also thank the families of patients whose generosity made this research possible.
This article includes supplementary materials available via the Internet at http://www.interscience.wiley.com/jpages/0364-5134/suppmat