Quantification of Circulating Plasma DNA in Friedreich's Ataxia and Spinocerebellar Ataxia Types 2 and 12

Abstract

DNA triplet repeat expansion-associated ataxias, Friedreich's ataxia, and different types of spinocerebellar ataxias (SCAs) are progressive multisystem neurodegenerative disorders. The diagnosis of this wide group of inherited ataxias is essentially based on clinical findings. Cell-free circulating DNA in plasma has been considered as a powerful tool in clinical diagnosis and prognosis of several human diseases. In the present study, clinically suspected patients were assessed on the International Co-operative Ataxia Rating Scale and further confirmed by molecular analysis of DNA triplet repeats. Quantification of plasma DNA using a highly sensitive and DNA-specific PicoGreen fluorescent assay was done. We found significantly high levels (p < 0.001) of plasma DNA of 167 ± 43 ng/mL in Friedreich's ataxia patients (n = 15), 148 ± 29 ng/mL in SCA2 patients (n = 10), and 137 ± 29 ng/mL in SCA12 patients (n = 25), whereas those of healthy controls (n = 20) was only 59 ± 15 ng/mL. Therefore, we were able to distinguish between ataxia patients and healthy controls using plasma DNA. Although the precise mechanism by which plasma DNA enters into circulation is not known, significantly higher concentrations of plasma DNA appears to be due to neuronal and muscular degeneration in these patients. Identification of genes in plasma DNA, which are overexpressed or novel, can be a promising tool for the prognosis of these diseases.

Quantitative Profiling and Identification of Plasma Proteins of Spinocerebellar Ataxia Type 2 Patients

“…Gene-specific PCR was carried out to confirm the number of CAG repeats in suspected SCA2 patients and in healthy controls as per our previous study [16]. The forward primer, SCA2-F flanked 177 bp upstream and reverse primer SCA2-R flanked 126 bp downstream to CAG repeats on ataxin-2 gene gave an amplification product of 303 ± 3n bp, where n is the number of CAG repeats.…”

Role of long purine stretches in controlling the expression of genes associated with neurological disorders

“…A well documented neurodegenerative disorder FRDA is identified with purine repeat in FXN gene ranging from 198-3600 (Swarup et al, 2011) and in rare cases, a repeat can be as long as 5100 (Wells, 2008). While the PR in healthy individuals is very low 60-102 (Swarup et al, 2011). Therefore, the aim of the present study was to look for uninterrupted PRs with repeat number, n ≥ 200 in genes and their association with neurological disorders.…”

“…Therefore, it directly depends on the length of PR, however, interruption by a single pyrimidine in the PR, destabilizes the triplex by many fold (Debin et al, 1999). A well documented neurodegenerative disorder FRDA is identified with purine repeat in FXN gene ranging from 198-3600 (Swarup et al, 2011) and in rare cases, a repeat can be as long as 5100 (Wells, 2008). While the PR in healthy individuals is very low 60-102 (Swarup et al, 2011).…”

In-vitro cytotoxicity of aflatoxin B1 to broiler lymphocytes of broiler chickens

“…The PicoGreen fluorescence assay measures the presence of double-stranded (ds) DNA, which is an indicative of cytotoxicity (Swarup et al, 2011). This assay was performed 24, 48 and 72 h after exposure, according to the protocol supplied by the manufacturer (Quant ItTM, Invitrogen).…”