Abstract |
Introduction: TDP-43 (TAR DNA-binding protein-43), encoded by the TARDBP gene, has
recently been identified as the molecular link, which connects the, until recently considered,
separate entities that currently form the FTD/ALS spectrum. Specifically, TDP-43 is the main
component of ubiquitinated inclusions found in both FTD and ALS. The direct causal
relationship of TDP-43 with the neurodegenerative process was suggested by the discovery
of pathogenic variants in the TARDBP gene, in families with members presenting with
FTD/ALS. Furthermore, TDP-43 proteinopathy has been observed in carriers of pathogenic
variants in additional genes associated with both FTD and ALS, as well as in patients with an
Alzheimer’s disease (AD) subtype, thus demonstrating a more general role of TDP-43 in the
neurodegenerative process.
The purpose of this dissertation is to investigate TDP-43 proteinopathy in Greek patients with
neurodegenerative diseases and in particular:
1. To measure TDP-43 protein levels in the cerebrospinal fluid (CSF) as a candidate
diagnostic biomarker for the FTD/ALS spectrum.
2. To detect genetic changes in the TARDBP gene, as well as in other genes associated
with FTD/ALS and to evaluate their contribution to the clinical phenotype.
3. To correlate genetic changes not only with the TDP-43 levels in the CSF, but also with
the levels of "classical" biomarkers for Alzheimer's disease (AD), i.e. total tau protein
(τΤ), its phosphorylated form (τΡ-181) and the β-amyloid peptide with 42 amino acids
(Αβ42).
Patients and Methods: The study population consisted of 54 patients with FTD, 61 patients
with ALS and 14 patients with FTD-ALS, who were diagnosed with the most recent diagnostic
criteria, supported by the “classical” AD biomarkers (τΤ, τΡ-181 and Αβ42), in the CSF, to rule out
the inclusion of patients with frontal and logopenic variant of AD in the FTD subgroup. The
quantitative determination of TDP-43 levels and “classical” biomarkers in the CSF was
performed by double-sandwich enzyme-linked immunosorbent assay (ELISA). Patients were screened for pathogenic hexanucleotide repeat expansion (GGGGCC)n in the
C9orf72 gene, using the repeat-primed polymerase chain reaction (PCR) method, while
C9orf72 (-) patients underwent whole exome sequencing.
The levels of biomarkers were compared with a control group of 28 healthy individuals
without mental or motor impairment and the genetic changes with an already very wellcharacterized
sample of 81 healthy aged adults from Crete. Also, 100 patients from Crete
diagnosed with dementia (of the AD type) were tested for pathogenic variants in FTD/ALS
genes.
Results: Statistically significant higher TDP-43 levels in the CSF were found in all patient
groups (FTD, ALS, FTD/ALS), compared to healthy controls. Patients with ALS showed a
tendency to have higher CSF TDP-43 levels compared to patients with FTD. Significantly higher
levels of τΤ were found in all groups, compared to healthy controls, with the FTD group
showing increased levels compared to the ALS group. The levels of τΡ-181 and Aβ42 did not
differ significantly among patient groups. ROC analysis showed that the combination of TDP-
43 with τ proteins, as expressed by the formula TDP-43 × τΤ/τΡ-181 has high sensitivity and
specificity (> 80%), for separation of ALS/FTD spectrum patients from healthy controls.
Genetic analysis revealed pathogenic or likely pathogenic variants in 18.6% (24/129) of
patients in the ALS/FTD spectrum. Genes harboring these variants included, in a decreasing
order of frequency, the C9orf72, TARDBP, GRN, VCP, SOD1 and FUS genes. The average age
of disease onset in patients with the C9orf72 repeat expansion was about 10 years earlier
compared to other patients. In 2 patients from Crete initially diagnosed as AD we identified
the p.Ile383Val variant.
Patients with ALS, either alone or concurrently with FTD, harboring the C9orf72 repeat
expansion and the MAPT p.Asp285As change had significantly lower levels of τΡ-181 compared
to the other patients. Also, carriers of rare APP variants had lower levels of Aβ42, compared
to non-carriers.
Conclusions: TDP-43 proteinopathy is a disease entity with significant clinical, biochemical
and genetic heterogeneity. It has been traditionally thought to be present in younger patients
compared to other neurodegenerative disorders; however, recently it is increasingly recognized among the elderly. The combination of TDP-43 with tau proteins in the CSF, as
expressed by the formula TDP-43×τT /τP-181, has high sensitivity and specificity and is therefore
a potential biomarker of TDP-43 proteinopathy. The most common gene variant associated
with TDP-43 proteinopathy in the Greek population is the C9orf72 repeat expansion with a
high frequency of 10,8% (14/129) compared to other European populations and is associated
with the disease onset 10 years earlier than the sporadic forms. The variant p.Ile383Val in the
TARDBP gene is another common pathogenic variant in the Greek population.
|