In collaboration with the research group of prof. Janez Plavec from the Institute of Chemistry in Ljubljana, we investigated the topology of various lengths of the antisense (G2C4) hexanucleotide repeat. In contrast with the current assumptions, we have shown that the different lengths of the G2C4 DNA sequences can form i-motifs and protonated hairpins in conditions similar to the physiological and not only in highly acidic conditions. Physiological conditions were simulated with a suitable pH, the addition of potassium, and 40% PEG. In additional experiments we have shown that the presence of the two complementary chains of the repeat DNA (G4C2 and G2C4) G-quadruplexes and i-motifs were preferably formed, but not double-stranded helix DNA as expected.
COBISS.SI-ID: 29086759
Using nuclear magnetic resonance and circular dichroism spectroscopy we show that DNA G4C2 with varying number of repeats d(G4C2)n form planar guanine quartets characteristic of G-quadruplexes. Additionally, we show DNA G-quadruplexes can form inter- and intra-molecularly in either parallel or anti-parallel orientation, based on d(G4C2) sequence length. This potential structural heterogeneity of longer disease-relevant repeats should therefore be taken into account when studying their role in disease pathogenesis.
COBISS.SI-ID: 27972647
An intronic GGGGCC (G4C2) hexanucleotide repeat expansion inC9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of G4C2 RNA can result in five different dipeptide repeat proteins (DPR: poly GA, poly GP, poly GR, poly PA, and poly PR), which aggregate into neuronal cytoplasmic and nuclear inclusions in affected patients, however their contribution to disease pathogenesis remains controversial. We show that among the DPR proteins, expression of poly GA in a cell culture model activates programmed cell death and TDP-43 cleavage in a dose-dependent manner. Dual expression of poly GA together with other DPRs revealed that poly GP and poly PA are sequestered by poly GA, whereas poly GR and poly PR are rarely co-localised with poly GA. Dual expression of poly GA and poly PA ameliorated poly GA toxicity by inhibiting poly GA aggregation both in vitro and in vivo in the chick embryonic spinal cord. Expression of alternative codon-derived DPRs in chick embryonic spinal cord confirmed in vitro data, revealing that each of the dipeptides caused toxicity, with poly GA being the most toxic. Further, in vivo expression of G4C2 repeats of varying length caused apoptotic cell death, but failed to generate DPRs. Together, these data demonstrate that C9-related toxicity can be mediated by either RNA or DPRs. Moreover, our findings provide evidence that poly GA is a key mediator of cytotoxicity and that cross-talk between DPR proteins likely modifies their pathogenic status in C9ALS/FTD.
COBISS.SI-ID: 30902823
This is an important study based on the cooperation of an international consortium that are part of the Project MinE. Project MinE aims to define the genetic component of ALS thrugh large scale GWAS and whole genome sequencing. Abstract: In the largest genome-wide association study in ALS that included 15,000 ALS patients and 27,000 controls, we detected differences in 6 genome regions that lead to an increase in the risk of ALS. By using an innovative approach a new locus on the chromosome 21 was identified that contains a hitherto undiscovered gene C21orf2 that was associated with a high risk of an onset of ALS. Additionally, two more loci that are associated with a higher incidence of ALS, MOPB and SCFD1, were also identified. This indicates that ALS is a complex genetic trait with a polygenic architecture. This study represents a stepping-stone for further research that might identify additional targets and shed new light on the onset of this disease.
COBISS.SI-ID: 3106220
Amyotrophic lateral sclerosis (ALS) is a complex fatal neurodegenerative disease characterized by progressive degeneration and loss of upper motor neurons in the cerebral cortex and lower motor neurons in brainstem and spinal cord. We established the frequencies of mutations in 4 major ALS-associated genes, SOD1, TARDBP, FUS, and C9ORF72 in a representative cohort of 85 Slovenian patients with sporadic form of ALS. Pathogenic massive hexanucleotide repeat expansion mutation in C9ORF72 was detected in 5.9% of patients and was the most common cause of the disease. In the remaining 3 genes, we identified 4 changes in 3 patients, p.Val14Met in SOD1, silent mutation p.Arg522Arg in FUS, and p.Gly93Cys in SOD1 together with a novel synonymous variant c.990A)G (p.Leu330Leu) in TARDBP gene, respectively. This study represents the first genetic screening of major causative genes for ALS in a cohort of sporadic ALS patients from Slovenia and is according to our knowledge the first such study in Slavic population. Overall, we genetically characterized 8.2% sporadic ALS patients.
COBISS.SI-ID: 31780313