Supervisors info:
Χρήστος Κρούπης, Αναπληρωτής Καθηγητής Κλινικής Βιοχημείας-Μοριακής Διαγνωστικής, Ιατρική Σχολή, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Μουτσάτσου-Λαδικού Παρασκευή, Καθηγήτρια Κλινικής Βιοχημείας- Ιατρικής Χημείας, Ιατρική Σχολή, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Ευρύκλεια Λιανίδου, Καθηγήτρια Αναλυτικής Χημείας-Κλινικής Χημείας, Τμήμα Χημείας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Summary:
Introduction: Frontotemporal dementia (FTD) is as common as Alzheimer’s disease at ages <65 years and concerns about 5-15% of the total dementia patients worldwide. It involves a number of clinic-pathologico-anatomical characteristics and major subtypes involve bvFTD (behavioral variant FTD), PPA (Primary progressive Aphasia) and FTD-MND (Motor Neuron Disease). The FTD spectrum also includes PSP (Progressive Supranuclear Palsy) and CBS (Cortico-basal Syndrome). The genetic basis of FTD is complex, it involves a number of genes such as GRN, MAPT, C9orf72, TARDBP etc. The TARDBP gene is located on chromosome 1 (1p36.22), consists of 6 exons and encodes the TDP-43 protein and has up to 6 different isoforms in humans, due to alternative splicing.
Its functions are mainly regulatory as it acts as a transcriptional repressor regulating mRNA splicing and metabolism during stress response states. TDP-43 is a DNA/RNA binding protein, it can bind both single or double stranded DNA/RNA sequences that contain GT/GU repeats. TDP-43 has been implicated in neurodegenerative disease (mainly ALS and FTD), and is a major constituent of ubiquitin-positive aggregates located in the cytoplasm (most commonly in the form of CTF-25kDa). TDP-43 belongs in the hn-RNP protein family consisting of two RNA recognition motifs (RRMs) followed by a glycine-rich C-terminal domain.
The aim of this study is the development of a novel, fast and accurate method for the detection of a single nucleotide polymorphism (SNP) p.Ile383Val (rs80356740, c.1147A>G, MAF: 0.002% EXac) in the TARDBP gene and its evaluation in Greek FTD patients so as to determine if it can be characterized as a founder mutation for the Greek FTD population.
Methods: Genomic DNA was isolated from the peripheral blood of 133 Greek FTD patients, after informed signed consent, and 60 healthy controls (all examined in the Attikon General University Hospital Neurology Clinic). A Real-Time qPCR protocol was designed using the LightCycler (Roche) instrument with allele specific hybridization probes and the results were evaluated using melting-curve analysis. The results were confirmed in a selected number of samples using Sanger DNA sequencing, and the BigDye Terminator v1.1 thermal cycling method, on the ABI310 genetic analyzer. The pathogenicity of the SNP was evaluated using various bionformatics tools.
Results: The method was rapid and robust and exhibited excellent PCR efficiency (E=2.06) on the melting curve analysis as well as reproducibility (Cq CV%< 3.5%) and specificity in genotyping. The melting temperature of the wild type allele was 55.26 ℃ whereas the mutant was 65.82℃ (CV%< 2.5%, ΔΤm=9,61). The melting-curve analysis of the Real-Time qPCR results for the p.Ile383Val in the TARDBP gene was reliable and detected 5 out of 133 FTD patients, in a heterozygous state, while in only one out of 60 controls. The in-silico tools yielded controversial results as SIFT, PolyPhen-2 and Align GVGD classified the SNP as tolerated while PhyloP, PhastCons and Mutation Taster classified the SNP as disease-causing since the area where it is located is highly conserved.
Conclusions: Out of the five patients found to be carriers of the SNP, three were diagnosed as FTD-SD (semantic FTD), one behavioral FTD and the last one belongs to the general spectrum of FTD. As a result, it is hypothesized that this SNP could correlate with the FTD-SD subtype. Furthermore, four out of five patients were men, this probably could indicate its importance in the male FTD population compared to women. The limited sample number as well as the low frequency of the SNP makes a definitive characterization impossible, although there are strong indications since the frequency in the European population is only 0.002% whereas it reaches 3.76% in Greek FTD patients, a remarkable increase. Further functional studies are required so as to characterize the pathogenicity of this DNA alteration.
Keywords:
Frontotemporal dementia, mutation, TARDBP, p.Ile383Val
