TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropathology: a genetic and histopathological analysis

Summary

Background

TDP-43 is a major component of the ubiquitinated inclusions that characterise amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitin inclusions (FTLD-U). TDP-43 is an RNA-binding and DNA-binding protein that has many functions and is encoded by the TAR DNA-binding protein gene (TARDBP) on chromosome 1. Our aim was to investigate whether TARDBP is a candidate disease gene for familial ALS that is not associated with mutations in superoxide dismutase 1 (SOD1).

Methods

TARDBP was sequenced in 259 patients with ALS, FTLD, or both. We used TaqMan-based SNP genotyping to screen for the identified variants in control groups matched to two kindreds of patients for age and ethnic origin. Additional clinical, genetic, and pathological assessments were made in these two families.

Findings

We identified two variants in TARDBP, which would encode Gly290Ala and Gly298Ser forms of TDP-43, in two kindreds with familial ALS. The variants seem to be pathogenic because they co-segregated with disease in both families, were absent in controls, and were associated with TDP-43 neuropathology in both members of one of these families for whom CNS tissue was available.

Interpretation

The Gly290Ala and Gly298Ser mutations are located in the glycine-rich domain of TDP-43, which regulates gene expression and mediates protein–protein interactions such as those with heterogeneous ribonucleoproteins. Owing to the varied and important cellular functions of TDP-43, these mutations might cause neurodegeneration through both gains and losses of function. The finding of pathogenic mutations in TARDBP implicates TDP-43 as an active mediator of neurodegeneration in TDP-43 proteinopathies, a class of disorder that includes ALS and FTLD-U.

Funding

National Institutes of Health (AG10124, AG17586, AG005136-22, PO1 AG14382), Department of Veterans Affairs, Friedrich-Baur Stiftung (0017/2007), US Public Health Service, ALS Association, and Fundació ‘la Caixa’.

Introduction

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease and is characterised by relentless degeneration of upper and lower motor neurons; this leads to progressive weakness and eventually death within 3–5 years.¹ Most cases are sporadic, but about 10% are familial.¹ Familial ALS is genetically heterogeneous: about 20% of cases are accounted for by mutations in Cu/Zn superoxide dismutase 1 (SOD1), which predominantly cause autosomal-dominant disease,² but other genes are mutated in rarer forms of ALS. These genes include senataxin (SETX), a DNA/RNA helicase that causes juvenile ALS,³ alsin (ALS2),4, 5 dynactin (DCTN1),6, 7 angiogenin (ANG),⁸ and synaptobrevin-associated membrane protein B (VAPB).⁹ The causes of sporadic ALS, which accounts for more than 90% of ALS cases, have been more difficult to identify genetically.

Mutations in a locus on chromosome 9p21, for which the gene has not yet been identified, are responsible for a form of ALS with frontotemporal lobar degeneration (FTLD).10, 11, 12 FTLD, a group of neurodegenerative disorders in which there is behavioural dysfunction, language dysfunction, or both, is thought to be the most common non-motor deficit in patients with ALS.12, 13, 14 There is accumulating evidence that frontal lobe dysfunction is present in up to 50% of patients with ALS, with as many as 20% showing abnormalities that meet Neary criteria for clinical FTLD.15, 16, 17

This clinical overlap between ALS and FTLD is particularly interesting because both ALS and the most common FTLD subtype, FTLD with ubiquitin inclusions (FTLD-U), are characterised by ubiquitin-positive, tau-negative, and α-synuclein-negative cytoplasmic inclusions in CNS neurons and glia. The major disease protein in both ALS and FTLD-U inclusions is the ubiquitinated TAR DNA-binding protein 43 (TDP-43).18, 19 TDP-43 pathology is not found in SOD1-associated familial ALS, suggesting that mechanistically ALS is heterogeneous.²⁰ TDP-43 is a 414-amino-acid nuclear protein encoded by TARDBP on chromosome 1p36.2. It was originally identified as a transcriptional repressor that binds to the TAR-DNA element of HIV and, because of its molecular weight of 43 kDa, it was named TDP-43.²¹ TDP-43 is involved in regulation of gene expression and splicing, and is part of a complex that splices the cystic fibrosis transmembrane conductance regulator gene (CFTR).22, 23, 24

The TDP-43 inclusions in ALS and FTLD-U put these two disorders in a class of neurodegenerative diseases where abnormal protein aggregation occurs.²⁵ Such disorders include Alzheimer's disease, Parkinson's disease, prion disorders, tauopathies, trinucleotide repeat disorders, and other rare brain amyloidoses. In all of these disorders, dominant mutations in the gene that encodes the deposited protein account for at least some cases of the disease. For example, mutations in the amyloid precursor protein gene APP, which encodes the Aβ peptide found in amyloid plaques in Alzheimer's disease, are a rare cause of this disease.²⁵ Likewise, mutations in the gene that encodes α-synuclein (SNCA), the major protein component of Lewy body deposits in Parkinson's disease, are a rare cause of this disease.²⁵ Mutations in MAPT, which encodes tau, the protein component of neurofibrillary tangles, cause some cases of frontotemporal dementia without ubiquinated TDP-43 deposits.²⁶ TARDBP is thus an obvious candidate gene for familial ALS and other familial frontotemporal dementia syndromes not caused by mutations in MAPT or progranulin. That several mutations in TARDBP were identified in ALS while this manuscript was in preparation is not surprising, although the previous studies27, 28 do not report an association with neuropathology. The mutations in these studies all cluster in exon 6 of TARDBP; two (Ala315Thr and Met337Val) segregate with disease in two familial ALS kindreds and two (Gln331Lys and Gly294Ala) were identified in patients with sporadic ALS. However, two other studies found no TARDBP mutations in 214 patients with FTLD,²⁹ 173 patients with FTLD,³⁰ or 237 patients with sporadic ALS.³⁰ Furthermore, no genetic association was seen between TARDBP polymorphic sites and FTLD or ALS,29, 30, 31 although there was a non-significant association with two single-nucleotide polymorphisms in patients with ALS and frontotemporal dementia in one study.³¹ Our aim was to further define the spectrum of TARDBP mutations by DNA sequence analysis of patients with ALS, FTLD, or both.