DNA AND CELL BIOLOGY Volume 30, Number 6, 2011 ª Mary Ann Liebert, Inc. Pp. 389–394 DOI: 10.1089/dna.2010.1165
Quantification of Circulating Plasma DNA in Friedreich’s Ataxia and Spinocerebellar Ataxia Types 2 and 12 Vishnu Swarup,1 Achal K. Srivastava,2 Madakasira V. Padma,2 and Moganty R. Rajeswari1
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 ¼ 15), 148 29 ng/mL in SCA2 patients (n ¼ 10), and 137 29 ng/mL in SCA12 patients (n ¼ 25), whereas those of healthy controls (n ¼ 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.
Introduction
F
riedreich’s ataxia (FRDA) and spinocerebellar ataxia (SCA) are cerebellar ataxias, which are progressive, multisystem, autosomal disorders with no effective treatment. Patients with these progressive ataxias manifest various neuronal complications and they are incapable of carrying out a daily routine. FRDA and SCA are primarily due to the presence of DNA triplet repeat expansion (TRE) located on different genes (Durr, 2010; Schmucker and Puccio, 2010). FRDA is an autosomal recessive ataxia, with a frequency of 1–2 persons in 30,000 (Campuzano et al., 1996). Based on clinical findings, FRDA is recognized by early age of onset (less than 25 years) with symptoms of gait and loss of limb coordination, blurred speech, sensory neuropathy, cardiac hypertrophy, and scoliosis (Harding, 1984; Campuzano et al., 1996). FRDA patients, in general (98%), are reported to have GAA repeats in the first intron of frataxin (fxn) gene, but 2% of FRDA patients have point mutations in the fxn gene (Campuzano et al., 1996; Babady et al., 2007). The repeat length of GAA can vary from 66 to 1200, but in rare cases the expansion can be as long as 1700 (Durr et al., 1996). Healthy controls carry 20–34 GAA repeats. FRDA is primarily due to insufficient production of frataxin protein. The deficiency of frataxin in FRDA patients is caused by the formation of
unusual triple helical DNA structures (Patel and Isaya, 2001; Jain et al., 2003), and DNA-RNA hybrid structures (Grabczyk et al., 2007) in the GAA expanded region and also due to heterochromatin formation (Saveliev et al., 2003) in the fxn gene. Frataxin, a mitochondrial protein, is present ubiquitously and plays a crucial role in iron homeostasis, storage of iron–sulphur clusters, and heme biosynthesis (Bencze et al., 2006). Therefore, the decreased expression of frataxin leads to oxidative stress in cells. SCAs are autosomal dominant cerebellar ataxias characterized by failure of muscle coordination due to degeneration of spinocerebellar tract. SCAs are either due to expansion of CAG repeats (e.g., SCA types 1, 2, 3, 8, 10, 12) or due to point mutations (e.g., SCA types 5, 13, 14, 27) in different genes on different chromosomes (Duenas et al., 2006). In SCA12 patients, the CAG repeats are expanded to 51–78 in the PPP2R2B gene (Srivastava et al., 2001; Holmes et al., 2002) (GenBank accession number: M64930). The PPP2R2B gene encodes a brain-specific regulatory subunit of the protein phosphatase PP2A. PP2A is a serine/threonine phosphatase and is widely expressed in neurons throughout the brain, including constitutive expression in Purkinje cells and the cerebellar cortex (Ferrigno et al., 1993). PPP2R2B regulates dephosphorylation activity of PP2A, which in turn regulates a wide array of cellular processes including cell growth and differentiation, DNA replication, long-term depression, and
Departments of 1Biochemistry and 2Neurology, All India Institute of Medical Sciences, New Delhi, India.
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390 apoptosis. However, the precise relationship between CAG repeat size and an altered/abnormal PPP2R2B transcript remains ambiguous (Strack et al., 1998; Holmes et al., 2003; Durr, 2010). However, SCA type 2 involves CAG expansion in the coding sequence of ataxin-2 gene. Symptoms of SCA2 include unsteady gait, slow saccades, abnormal eye movements with frequent neuropathy, chorea dystonia, and dementia (Imbert et al., 1996). The CAG repeat length in SCA2 patients varies from 34 to 55. Ataxin-2, the mutant protein in SCA2, interacts with polyA-binding protein 1 and plays an important role in RNA metabolism. Cerebellar Purkinje neurons are the first target of this mutated protein. Cell-free circulating plasma DNA is found in blood and other body fluids such as urine and amniotic fluid. Plasma DNA is present in very low quantities in healthy controls. However, elevated levels are reported in a number of diseases and infections (Annemarie et al., 2002; Swarup and Rajeswari, 2007). Therefore, cell-free DNA in plasma has emerged as an attractive tool in the early prognosis of several cancers such as head and neck carcinoma (Nawroz et al., 1996), breast cancer (Shao et al., 2002; Catarino et al., 2008), and lung cancer (Tsou et al., 2002; Kumar et al., 2010) and also other diseases such as stroke (Rainer et al., 2003) and myocardial infarction (Chang et al., 2003). Although the mechanism of origin of plasma DNA is not yet clear, it has left several landmarks in human disorders. It has been documented that blood–brain barrier (BBB) is disrupted in Alzheimer’s disease (Caserta et al., 1998) and stroke (Kidwell et al., 2008). The purpose of the present study was to assess the plasma DNA levels in FRDA and SCA types 2 and 12 patients against healthy controls. Materials and Methods Clinical samples Blood samples were collected from clinically suspected patients: 62 SCA12, 32 SCA2, and 22 FRDA from the Ataxia Clinic, Department of Neurology, All India Institute of Medical Sciences, New Delhi, India. Twenty healthy volunteers were also included in the present study. The present work was carried out with consent of patients and healthy controls and as per Institutional Ethical Committee guidelines (Ethical Clearance No. A-52/9.8.2006). Molecular analysis of DNA triplet repeats The following primers were used for analysis of DNA repeats: for SCA2, SCA2-F 50 -GTCCTTCTCCCCCTCGCCA30 as forward primer and SCA2-R 50 -CACCGAGGAGG GAGCCGTG-30 as reverse primer to amplify the CAG stretch on the ataxin-2 gene on 12th chromosome (Ivan and Forrest, 1998). PCR was run in a 20 mL mixture containing 1 buffer, 0.2 mM dNTP, 3 mM MgCl2, 30 pmol SCA2-F/SCA2-R, 10% dimethyl sulfoxide, 1 U Taq polymerase (Fermentas), and 40 ng genomic DNA under 30 cycles of 968C for 1 min, followed by 658C for 1 min and 728C for 2 min, with a final extension at 728C for 10 min. Primers used for gene amplification in SCA12 are forward primer 50 -TGCTGGGAAA GAGTCGTG-30 and reverse primer 50 -AGGATTCAGGCTT GCCT-30 (Holmes et al., 1999). Amplification was done using PCR buffer, 0.2 mM dNTPs, 3 mM MgC12, 20 pmol SCA12-F/SCA12-R, 1 U Taq polymerase (Fermentas), and
SWARUP ET AL. 80 ng genomic DNA under the following conditions: 30 cycles comprised of 968C for 1 min, 568C for 40 sec, and 728C for 1 min, with a final extension at 728C for 10 min. The PCR products were visualized on a 3% ethidium bromide-stained agarose gel using UV irradiation, and allele size was determined by comparison with 50-bp DNA ladder. Number of CAG repeats for SCA12 and SCA2 was further reconfirmed by sequencing (data not shown). To analyze GAA repeats in fxn gene in healthy individuals and FRDA patients, we used the following primers: FRDA-F 50 -GGAGGGA TCCGTCTGGGCAAAGG-30 as forward primer and FRDA-R 50 -CAATCCAGGACAGTCAGGGCTTT-30 as reverse primer in the long-range PCR (Lamont et al., 1997). Amplification was done using Phusion High-Fidelity PCR Kit (Finnzymes) containing 1 HF-PCR buffer, 0.2 mM dNTPs, 20 pmol each FRDA-F/FRDA-R, 1 U high-fidelity (HF) Taq polymerase, 100 ng genomic DNA. The long-range PCR protocol comprised of 20 cycles of following steps: 948C for 20 s, 688C for 2.5 min, followed by 17 cycles in which the length of the 688C step was increased by 15 s/cycle. The PCR product (20 ml) was visualized on a 1.2% ethidium bromide-stained agarose gel using UV irradiation, and allele size was determined by comparison with 1-kb DNA ladder. PCR amplification for all the samples was done twice on an MJ mini cycler (BioRad). PicoGreen fluorescent assay Plasma DNA was isolated from 200 mL of filtered plasma from healthy control and genetically confirmed patients. The PicoGreen (Molecular Probes) assay was done according to protocol supplied by the manufacturer. PicoGreen dye was diluted 1:200 with TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.5) and incubated with plasma DNA in the dark at room temperature for 5 min. To minimize photobleaching effects, time for fluorescence measurement was kept constant for all samples. All the fluorescence measurements were recorded on a Biotek spectrofluorometer FLx800 (Biotek) with the help of a multidetector microplate reader. Fluorescence emission of PicoGreen alone (blank) and PicoGreen with DNA were recorded at 528 nm using an excitation wavelength of 485 nm at room temperature (258C). A standard curve was generated using double-stranded (ds) lambda DNA provided by the manufacturer. The fluorescence emission measurement of each plasma DNA sample was an average of three independent measurements and the corresponding standard deviation is indicated in the data. SPSS v11.5 was used for statistical analysis. Differences between two groups (control vs. patients) were evaluated using Mann–Whitney U test. Statistical significance was set at p < 0.05. Results All clinically suspected patients were assessed by International Co-operative Ataxia Rating Scale (ICARS) (Trouillas et al., 1997) (Table 1). We recruited 62 patients suspected of having SCA12, 32 with SCA2, and 22 with FRDA from the Ataxia Clinic. These patients were the total number of patients enrolled during the period of a 3-year study (2007– 2010). ICARS score gives a good correlation with severity of ataxia. The higher the ICARS score, the more severe the ataxia. ICARS scores (mean value standard deviation) were 48 11 (range: 32–56), 41 13 (range: 33–62), and 38 12
GAA CAG CAG n is the number of subjects in each group. SD, standard deviation.
53 43 55
0.5–12 0.5–8 0.5–16 4–22 2–27 30–62 Friedreich’s ataxia 13 5 Spinocerebellar ataxia 2 21 9 Spinocerebellar ataxia 12 49 9
48 11 32–56 41 13 33–62 38 12 24–69
Mean SD Type Range of repeats Range Mean SD
Mean SD
Number of repeats
DNA triplet repeats
Range
Molecular findings
ICARS score
Range
Although the total number of patients (who were admitted in hospital or visited the outpatient department) were suspected of having ataxias and other movement disorders is very high, only those patients who showed characteristic symptoms of FRDA, SCA2, and SCA12 disorders were further tested for TRE. Selecting symptomatic patients was the main reason of getting such a large patient pool of these diseases, and ICARS scoring of all these suspected patients has clearly indicated the severity of disease. Therefore, we were able to confirm 15 patients with FRDA, 10 patients with SCA2, and 25 patients with SCA12. PCR analysis of SCA2 patients using the forward primer 177 bp upstream and reverse primer 126 bp downstream to CAG repeats on ataxin-2 gene gave an amplification product of 303 þ 3N bp, where N
Mean SD
Discussion
Duration of disease (years)
Quantification of plasma DNA was done using fluorescent PicoGreen dye, which is highly specific for dsDNA. PicoGreen alone has very low emission intensity at 528 nm (excitation at 485 nm); however, it gives a very high intensity at 528 nm on binding to dsDNA. Interestingly, the interference from ssRNA and ssDNA in the estimation of dsDNA by PicoGreen assay is negligible as the complexes of PicoGreen with ssRNA or ssDNA gives a minimum fluorescence (Molecular Probes). We found 167 43 ng/mL (range: 64– 703 ng/mL) in FRDA patients, 148 29 ng/mL (range: 110– 200 ng/mL) in SCA2 patients, and 137 29 ng/mL (range: 111–211 ng/mL) in SCA12 patients. The concentration of plasma DNA in healthy control was found to be 59 15 ng/ mL (range: 40–94 ng/mL) (Fig. 2).
Age of patients at onset (years)
Quantification of plasma DNA
Clinical findings
Genetic analysis using long-range PCR confirmed 15 patients carrying expanded GAA triplets in both alleles out of 22 clinically suspected FRDA patients. The range of allele expansion in all patients was from 3 to 5 kb. A clear demarcation of two bands of 3 kb on agarose gel corresponding to >550 GAA repeats confirmed the FRDA disease of the patient (Fig. 1a, lane 5). The mean of GAA triplet repeats in the larger allele was 1181 117 (range: 870–1220) in confirmed patients. Moreover, we also found three heterozygous patients carrying one normal and one expanded allele (Fig. 1a, lane 4). As SCA2 and 12 are autosomal dominant phenotypes, these were confirmed by the presence of a single allele carrying expanded CAG repeats in their respective genes. Genetic analysis of 32 clinically suspected SCA2 patients revealed that only 10 patients carried expanded CAG repeats in the range of 36–42 (mean 41 13) (Figure 1c, Lane 1; Table 1). The ataxin-2 gene in healthy individuals had 22–28 CAG repeats (Fig. 1c, lane 2). The mean number of CAG repeats was 62 6 (range: 53–77) in SCA12 patients (Fig. 1b, lanes 2 and 4), and 25 patients were confirmed to contain these abnormal expanded repeats when compared with 9–11 CAG repeats in healthy controls (Lane 1, Fig. 1b).
Table 1. Clinical Parameters, Genetic Analysis, and Plasma DNA of Friedreich’s Ataxia (n ¼ 15), Spinocerebellar Ataxia 2 (n ¼ 10), and Spinocerebellar Ataxia 12 (n ¼ 25) Patients and Healthy Controls (n ¼ 20)
Genetic analysis of TRE
Mean SD
Plasma DNA (ng/mL)
(range: 24–69) for FRDA, SCA2, and SCA12 patients, respectively. Mean age of patients was 13 5, 21 9, and 49 9 years for FRDA, SCA2, and SCA12 suspected patients (Table 1).
1181 117 (Controls, 21 2) 870–1220 (20–25) 167 43 (59 15) 64–703 (40–94) 40 2 (Controls, 23 3) 36–42 (22–28) 148 29 (59 15) 110–200 (40–94) 62 6 (Controls, 9 1) 53–77 (9–11) 137 29 (59 15) 111–211 (40–94)
391
Range
PLASMA DNA IN FRDA AND SCA
392
SWARUP ET AL.
FIG. 1. Representative gel photographs of (a) Friedreich’s ataxia: 1.2% agarose gel electrophoresis of DNA samples: lane M, 1 kb DNA ladder; lane 1, positive control l-DNA with 1.3 kb primer set; lane 2, healthy control; lanes 3 and 6, clinically suspected patients but genetic test proved them normal for GAA repeats; lane 4, heterozygous patient with 1.4 and 4 kb bands; lane 5, homozygous patient with 4 and 3 kb bands. (b) Spinocerebellar ataxia 12 (SCA12): 3% agarose gel: lane M, 50 bp DNA ladder; lane 1, healthy control; lanes 2 and 4, patient; lane 3, clinically suspected SCA12 patient but found to be normal for CAG repeats. (c) SCA2: 3% agarose gel of DNA: lane M, 50 bp DNA ladder; lane 1, SCA2 patient; lane 2, healthy control.
is the number of CAG repeats (Ivan and Forrest, 1998). Therefore, the PCR product of the healthy individual with N ¼ 20 CAG repeats produced 384 bp (303 þ 27 3 bp) band (Lane 2, Fig. 1b). Of 32 patients who were clinically suspected, only 10 were confirmed as having SCA2. The smallest pathogenic allele contained 42 CAG repeats in contrast to the normal allele with 26 repeats. Hence, the difference between pathogenic and normal alleles was quite large and we were easily able to confirm the number of triplets in these diseases. Similarly, forward primer SCA12-F flanked 79 bp upstream from the 50 end, and reverse primer SCA12-R was located 171 bp downstream from the 30 end of the CAG stretch on the PPP2R2B gene. Amplification using this primer set produced a constant region of (230 þ 3N bp). Here also, a clear demarcation was seen between pathogenic allele and normal allele. In SCA12 patients, the minimum length of pathogenic allele was 53 CAG repeats, whereas the normal allele of healthy individuals has 17 repeats. However, in case of FRDA, presence of two expanded alleles confirmed the
FIG. 2. Plasma DNA in healthy controls and Friedreich’s ataxia (FRDA), SCA2, and SCA12 patients. Black line represents the median value. *p < 0.001, Mann–Whitney U test.
homozygous status of the patients. Further, the difference between normal and pathogenic allele was significantly large, 1.4 kb (including 20 GAA repeats) and 3 kb (550 GAA repeats), respectively, and could easily differentiate between healthy persons and FRDA patients. Long-range PCR was very successful in amplifying extremely long GAA repeats even up to 1400 repeats (Fig. 1a). Moreover, heterozygous patients were also well identified with the help of long-range PCR. The presence of one high molecular size band and one normal size band on agarose gel confirms the heterozygous status. Of 22 clinically suspected patients, we confirmed 15 homozygous and 3 heterozygous FRDA patients. However, these heterozygous patients were not included for quantification of plasma DNA as the primary cause of the FRDA in them was a point mutation in normal allele and not the GAA triplet expansion (Campuzano et al., 1996; Lamont et al., 1997). Conventionally, DNA can be detected by a variety of DNA-binding molecules. For example, ethidium bromide is widely used to detect and visualize the DNA in PCR products. Several other compounds, such as acridine orange, Hoechst-33258, and methylene blue, also bind to DNA ( Jain et al., 2003; Singhal and Rajeswari, 2010). However, PicoGreen is highly specific and sensitive for dsDNA (upto 25 pg/mL). Moreover, the linear detection range of the PicoGreen assay in a standard fluorometer extends over more than 4 orders of magnitude in DNA concentration from 25 pg/mL to 1000 ng/mL with a single dye concentration (Molecular Probes, Eugene). The sensitivity of PicoGreen dye is not affected by the presence of contaminants that are common in DNA preparations, including salts, urea, ethanol, chloroform, detergents, proteins, etc. Therefore, DNA quantification using PicoGreen is more reliable (Schofield, 2004; Chiminqgi et al., 2007). The plasma DNA was found to be significantly high ( p < 0.001, Mann–Whitney U test: 167 43 ng/mL (range: 64–703 ng/mL) in FRDA patients, 148 29 ng/mL (range: 110–200 ng/mL) in SCA2 patients, and 137 29 ng/mL (range: 111–211 ng/mL) in SCA12 patients when compared with 59 15 ng/mL (range: 40–94 ng/ mL) in healthy controls (Fig. 2). Surprisingly, comparison of the ICARS scores of FRDA, SCA2, and SCA12 with their respective plasma DNA concentration did not show any
PLASMA DNA IN FRDA AND SCA significant correlation (Spearman rank correlation coefficient: r ¼ 0.15, p ¼ 0.5 for SCA12; r ¼ 0.37, p ¼ 0.3 for FRDA; and r ¼ 0.52, p ¼ 0.3 for SCA2 patients). Scalzo et al. (2009) also did not find any marked difference between plasma DNA and clinical parameters in Parkinson’s disease. High concentrations of plasma DNA in FRDA (by *3-folds, p < 0.001) and SCA2 and 12 (by *2.5-folds, p < 0.001) patients indicate that the DNA fragments originated from neuronal breakdown and muscular atrophy. Similarly, in breast cancer (105 ng/mL) (Shao et al., 2002), gastric cancer (205 ng/mL) (Kolesnikova et al., 2008), myocardial infarction (511 398 ng/mL) (Chang et al., 2003), lung cancer (157 mg/L) (BeauFaller et al., 2003), and systemic lupus erythematosus (4,024 ng/mL) (Raptis and Menard, 1980), cellular breakdown has been indicated. Metastasis, hypoxia, and necrosis may play a critical role in raising the levels of circulating DNA in cancer patients. However, the actual cause for high levels of plasma DNA in the patients’ circulation is yet unknown. The compromised blood–brain barrier could have some role in releasing the plasma DNA in circulation. In conclusion, our data suggest that plasma DNA has the potential to be used as an easy prognostic marker in triplet repeat-associated neurological disorders. Acknowledgments Financial assistance from the Indian Council of Medical Research of India (5/4-5/5/Neuro2006/NCD-I) is gratefully acknowledged. V. Swarup thanks the Council of Scientific and Industrial Research for providing Senior Research Fellowship (9/6(328)/2005-EMR-I). Disclosure Statement No competing financial interests exist. References Annemarie, Z., Zangemeister-Wittke, U., and Stahel, R.A. (2002). Circulating DNA: a new diagnostic gold mine? Cancer Treat Rev 28, 255–271. Babady, N.E., Carelle, N., Wells, R.D., Rouault, T.A., Hirano, M., Lynch, D.R., Delatycki, M.B., Wilson, R.B., Isaya, G., and Puccio, H. (2007). Advancements in the pathophysiology of Friedreich’s ataxia and new prospects for treatments. Mol Genet and Metabol 92, 23–35. Beau-Faller, M., Gaub, M.P., Schneider, A., Ducrocq, X., Massard, G., and Gasser, B. (2003). Plasma DNA microsatellite panel as sensitive and tumor-specific marker in lung cancer Patients. Int J Cancer 105, 361–370. Bencze, K.Z., Kondapalli, K.C., Cook, J.D., and McMahon, S. (2006). The structure and function of frataxin. Crit Rev in Biochem and Mol Biol 41, 269–291. Campuzano, V., Montermini, L., Molto`, M.D., Pianese, L., Cosse´e, M., Cavalcanti, F., Monros, E., Rodius, F., Duclos, F., Monticelli, A., Zara, F., Can˜izares, J., Koutnikova, H., Bidichandani, S.I., Gellera, C., Brice, A., Trouillas, P., Michele, G.D., Filla, A., Frutos, R.D., Palau, F., Patel P.I., Donato, S.D., Mandel, J., Cocozza S., Koenig M., and Pandolfo, M. (1996). Friedreich’s ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 271, 1423– 1427. Caserta, M.T., Caccioppo, D., Lapin, G.D., Ragin, A., and Groothuis, D.R. (1998). Blood–brain barrier integrity in Alzheimer’s
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Address correspondence to: Moganty R. Rajeswari, M.Sc., Ph.D. Department of Biochemistry All India Institute of Medical Sciences New Delhi 110029 India E-mail:
[email protected] Received for publication October 23, 2010; received in revised form December 4, 2010; accepted December 8, 2010.
