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

Abstract

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'.

Figure 1. FALS pedigrees

(A). Family ND654 with TARDBP p.Gly290Ala (G290A) and (C). QBB with p.Gly298Ser (G290S). The sequencing electropherograms with heterozygous peaks marked with a red arrow are shown in B and D, respectively. Each individual is marked with generation and individual numbers. The proband in each family is marked with an arrowhead. Gender was removed from many individuals (diamonds) to protect privacy. Multiple siblings are indicated with a number inside the symbol. Clinical status is in the legends and TARDBP genetic testing results are indicated at upper right the symbol as “Neg” (negative) or with the mutation. OC: obligate carrier.

Figure 1. FALS pedigrees

(A). Family ND654 with TARDBP p.Gly290Ala (G290A) and (C). QBB with p.Gly298Ser (G290S). The sequencing electropherograms with heterozygous peaks marked with a red arrow are shown in B and D, respectively. Each individual is marked with generation and individual numbers. The proband in each family is marked with an arrowhead. Gender was removed from many individuals (diamonds) to protect privacy. Multiple siblings are indicated with a number inside the symbol. Clinical status is in the legends and TARDBP genetic testing results are indicated at upper right the symbol as “Neg” (negative) or with the mutation. OC: obligate carrier.

Figure 2. Immunohistochemical detection of TDP-43 inclusions in Family QBB with p.Gly298Ser

TDP-43 immunohistochemistry conducted in cases II-7 (A, C, E) and III-4 (B, D, F) using a rabbit polyclonal antibody to TDP-43 (Protein Science, Chicago, IL). Filamentous and round TDP-43 immunopositive inclusions were observed in anterior horn cells (A, B, C) of the spinal cord and in substantia nigra neurons (D) of both cases. Additional TDP-43 inclusions were detected in the cingulate gyrus of case II-7 (E) and in the amygdala of both cases (F).

Figure 3. Other examples of TDP-43 pathology

Other TDP-43 positive lesions included dense round inclusions in the dentate gyrus of the hippocampus in case III-4 (A); ring-like dense cytoplasmic inclusions (thin arrows) and diffuse stippled cytoplasmic staining with nuclear clearing or “pre-inclusions” (arrowheads) in the entorhinal cortex (B), temporal cortex (C), hyppoglossal nucleus (D), substantia nigra (E), and amygdala (F), as well as a glial inclusions (thick arrow, C). All images obtained from case III-4, except for C, from case II-7.