Journal of the Neurological Sciences 341 (2014) 41–45
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Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Role of glutathione S-transferases in the spinocerebellar ataxia type 2 clinical phenotype D. Almaguer-Gotay ⁎, L.E. Almaguer-Mederos, R. Aguilera-Rodríguez, A. Estupiñán-Rodríguez, Y. González-Zaldivar, D. Cuello-Almarales, J.M. Laffita-Mesa, Y. Vázquez-Mojena Center for the Research and Rehabilitation of Hereditary Ataxias—CIRAH, Cuba
a r t i c l e
i n f o
Article history: Received 11 September 2013 Received in revised form 21 March 2014 Accepted 24 March 2014 Available online 1 April 2014 Keywords: Oxidative stress Polyglutamine disease Spinocerebellar ataxia type 2 Glutathione S-transferase Antioxidant enzymes Presymptomatic
a b s t r a c t Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative and incurable hereditary disorder caused by a CAG repeat expansion mutation on ATXN2 gene. The identification of reliable biochemical markers of disease severity is of paramount significance for the development and assessment of clinical trials. In order to evaluate the potential use of glutathione-S-transferase (GST) activity as a biomarker for SCA2, a case–control study in 38 affected, presymptomatic individuals or healthy controls was conducted. An enlarged sample of 121 affected individuals was set to assess the impact of GST activity on SCA2 clinical expression. There was a significant increase in GST activity in affected individuals relative to controls, although sensibility and specificity were not high. GST activity was not significantly influenced by sex, age, disease duration or CAG repeat size and did not significantly influence disease severity markers. These findings show a disruption of in vivo GST activity in SCA2, suggesting a role for oxidative stress in the neurodegenerative process. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
2. Methods
Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative hereditary disorder caused by a CAG repeat expansion mutation on the first exon of ATXN2 gene. It has been widely characterized in its clinical, neurophysiological, molecular and epidemiological aspects [1,2]. However, further studies are needed for the identification of biochemical biomarkers of disease severity and progression. Several lines of evidence suggest glutathione S-transferase activity as a candidate biomarker in SCA2. Human glutathione S-transferases (GSTs, EC 2.5.1.18) are involved in the metabolism of xenobiotics, protection against oxidative stress and prevention of apoptosis [3]. Coincidentally, SCA2 has been linked to oxidative stress, particularly to altered activity of key antioxidant enzymes [4,5], and to increased apoptosis [6]. Therefore, it is expected that the GST activity being altered in SCA2 mutation carriers as a response to the neurodegenerative process is caused by the expression and dysfunction of mutant ataxin-2. The largest Cuban SCA2 population was studied in order to demonstrate the potential value of GST activity as a biomarker for SCA2. The association between serum GST activity and expanded CAG repeat size, age at onset, disease duration, disease severity score and disease progression rate was also assessed.
2.1. Subjects
⁎ Corresponding author at: Libertad No. 26. Entre 12 y 14. Rpto: El llano, Holguín CP: 80100, Cuba. Tel.: +53 24 462823. E-mail addresses: [email protected], [email protected] (D. Almaguer-Gotay).
http://dx.doi.org/10.1016/j.jns.2014.03.045 0022-510X/© 2014 Elsevier B.V. All rights reserved.
A case–control study was conducted to assess a possible association between GST activity and SCA2. Thirty-eight molecularly confirmed SCA2 affected individuals (11 men and 27 women), aged 17 to 62 years, were recruited at the Center for the Investigation and Rehabilitation of Hereditary Ataxias, Holguín, Cuba. Age and sex matched groups of presymptomatic individuals (aged 18 to 66 years) and healthy controls (aged 22 to 64 years) were included in the study. Affected and presymptomatic subjects were considered separately or as a single group (“mutation carriers”). The sample of affected individuals was enlarged to 121 subjects (aged 17 to 68 years) to evaluate the contribution of GST activity to the age at onset variability. Informed consent was obtained from the affected, presymptomatic and control individuals.
2.2. Clinical assessment A complete neurological examination was conducted and the clinical diagnosis of SCA2 was based on positive family history and disease manifestation such as ataxic gait, dysarthria, dysmetria, dysdiadochokinesia and slowing of saccadic eye movements. The age at onset was defined as the onset of motor impairment, and disease duration as the years from onset of clinical expression to the last examination. Disease severity was assessed using the Scale for the Assessment and Rating of Ataxia (the
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D. Almaguer-Gotay et al. / Journal of the Neurological Sciences 341 (2014) 41–45
SARA) [7]. The disease progression rate at presentation was measured as the SARA score/disease duration.
3. Results 3.1. Association of GST activity with SCA2
2.3. CAG repeat size determination Genomic DNA was isolated from peripheral blood leukocytes using a standard protocol. The ATXN2 CAG repeat length was assessed by polymerase chain reaction (PCR) and the amplification with the previously published UH10 and UH13 oligonucleotide primers [8]. Aliquots of the PCR products were then sized by fragment analysis on an ALF Express II apparatus (Amersham Biosciences, Sweden) in comparison to internal and external size markers using Allele Links 1.0 software (Amersham Biosciences, Sweden). Cases with 32 or more repeats were designated as ATXN2 gene carriers in accordance with published association with disease expression [9].
2.4. GST activity measurement Serum was obtained by blood centrifugation at 3000 g for 5 min. It was frozen immediately after and stored at −20 °C until analyses. GST activity was measured by a spectrophotometric method as described by Habig [10]. Briefly, GST activity was determined using 25 mmol/L 1-chloro-2,4-dinitrobenzene (CDNB) and 20 mmol/L reduced glutathione (GSH) as cosubstrates in 0.5 mol/L potassium phosphate buffer (pH 6.5). The reaction was monitored at 340 nm using a BioMate spectrophotometer (ThermoSpectronic, USA).
In the full data of affected, presymptomatic and control individuals, GST activity varied between 6.77 and 18.75 IU/L, with a mean of 10.66 ± 2.39 IU/L. GST activity did not correlate significantly with the age (r = −0.072; p = 0.43). There was a tendency for females to have a higher GST activity (10.95 ± 2.40 IU/L) relative to males (10.02 ± 2.24 IU/L); however, this difference failed to reach statistical significance (t = −1.87; p = 0.064). The composition by sex, age and CAG repeat size of the studied groups is shown in Table 1. There were no significant differences according to age for these three groups; however, affected individuals showed significantly higher CAG repeat lengths than presymptomatic individuals (p b 0.001). Mutation carriers showed an increased GST activity in comparison to controls, which is due to a highly significant increase in GST activity in affected individuals (11.9 ± 2.24 IU/L). Actually, presymptomatics showed an increased GST activity (10.3 ± 2.11 IU/L) in comparison to the controls (9.77 ± 2.33 IU/L) but the difference was not significant (Fig. 1A and B). The age showed no significant correlation with GST activity in any studied group (p N 0.05). Control females showed a significantly higher GST activity relative to control males (p b 0.001); this finding was not replicated in affected or presymptomatic individuals (p N 0.05). As presymptomatic patients represent a transition state between healthy and affected individuals, the impact of increasing the risk to become affected on the GST activity was assessed. Although there was a tendency for GST activity to increase with the increase in the risk to become affected, no significant differences were found between individuals belonging to different risk classes (F = 0.017; p = 0.98) (Fig. 1C).
2.5. Statistical analysis Descriptive statistics were used to describe central tendencies and dispersion of the variables under study. A normal distribution for the variables was verified using the Shapiro–Wilk test. Comparison of GST activity between mutation carriers (clinically affected subjects plus presymptomatic individuals) and control individuals was performed by means of the Student's t-test. This test was also used for comparing GST activity between sexes. One-way ANOVA was used as a means of comparison in the circumstances when more than two groups were involved. Tercils were defined according to frequency distributions for the risk to become affected in presymptomatic individuals, age at onset and residual age at onset after adjustment by linear regression for the expanded CAG repeat number in affected subjects. The risk to become affected was calculated by survival analysis according to the CAG repeat number and current age for each presymptomatic individual as previously described [11]. Correlations between GST activity, age, age at onset, disease duration and expanded CAG repeat were assessed by Pearson's correlation test, once they were considered as parametric variables. Statistical significance was defined as p b 0.05. All statistics were computed in SPSS software (version 15.0) [12]. Positive predictive values, negative predictive values, sensitivity, specificity, likelihood ratios and odds ratio were calculated using EpiMax algorithm [13].
3.2. Sensitivity and specificity GST activity at different cut-off values was analyzed to determine its sensitivity and specificity for diagnosing SCA2 (Table 2). The odds ratio to be diagnosed with SCA2 for a GST activity value of 11 IU/L was 4.21 with a specificity of 71.1 and a sensitivity of 63.2. This would indicate that a false-positive rate for this potential diagnostic biomarker within the general population would be 31.4% (a positive predictive value of 68.6) and that 34.1% (a negative predictive value of 65.9%) of those individuals tested that are likely to have SCA2 would be falsely detected as normal. Predictive values were not better for a cut-off value of 13 IU/L. 3.3. Significance of GST activity for the SCA2 clinical phenotype In order to get insight into the significance of GST activity at the age of onset, an enlarged sample of 121 affected patients was studied. In this data, sex did not significantly influence on clinical or molecular SCA2 markers, nor on GST activity (Table 3). Also, GST activity was not significantly influenced by age, disease duration or CAG repeat number (p N 0.05), and had no influence on the age at onset (F = 1.095; p = 0.34), even after adjustment for the expanded CAG repeat number (F = 0.335; p = 0.72) (Fig. 2).
Table 1 Descriptive statistics for general variables included in the study.
Sex (male/female) Age (years) (Mean ± SD) Expanded CAG repeat number (Mean ± SD) Non-significant differences (ns). a One-way ANOVA. b Student's t-test; p N 0.05.
Affected (n = 38)
Presymptomatics (n = 38)
Controls (n = 38)
p
11/27 38.7 ± 10.09 42.7 ± 4.73
11/27 37.7 ± 10.49 36.8 ± 2.26
11/27 37.4 ± 9.77 –
– 0.86a(ns) b0.001b
D. Almaguer-Gotay et al. / Journal of the Neurological Sciences 341 (2014) 41–45
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Fig. 1. Glutathione-S-transferase activity by clinical status. A) Comparison of GST activity between mutation carriers (affected + presymptomatics) and healthy subjects. A significant increase in GST activity was noted for mutation carriers related to the controls (**p = 0.005). B) Comparison of GST activity between affected, presymptomatic and healthy subjects. A significant increase in GST activity was noted for affected individuals related to presymptomatic and control individuals (***p b 0.001; **p = 0.005). C) GST activity in presymptomatic individuals according to the risk of becoming affected. A non-significant increase in GST activity as the individual's risk advances was obtained (p N 0.05).
4. Discussion
In a subsample of 50 affected individuals it was possible to obtain the SARA and disease progression rate scores. The SARA total score was not significantly influenced by sex or age (p N 0.05), but it showed significant associations with age at onset, disease duration and CAG repeat number. In addition, GST activity had a statistically significant but low impact on the SARA total score (Fig. 3). At multiple linear regression only disease duration and the CAG repeat number were significant predictors of the SARA total score (R2 = 0.45). Similarly, disease progression rates were not significantly influenced by sex (p N 0.05) but it showed significant associations with age at onset, CAG repeat number and GST activity (Fig. 3). However, at multiple linear regression only age at onset and the CAG repeat number were predictors of disease progression rates (R2 = 0.74).
Human glutathione S-transferases are widely expressed and provide the most important line of cellular defense against electrophilic genotoxic endogenous compounds and xenobiotic chemicals. They are also involved in the protection against oxidative stress and prevention of apoptosis [3, 14,15]. The present study showed that increased GST activity is associated to SCA2, probably even since the presymptomatic stage. In fact, the affected and presymptomatic individuals showed 21.8% and 5.5% increases in GST activity, respectively, relative to age and sex matched controls. However, GST activity did not show high sensitivity and specificity; therefore, its utility for diagnostic purposes is very limited.
Table 2 Positive predictive value (PPV), negative predictive value (NPV), sensitivity, specificity, likelihood ratio (LR), odds ratio (OR) and p-values for GST activity. Affected vs. controls
PPV (%)
NPV (%)
Sensitivity (%)
Specificity (%)
LR
OR (95% CI)
p
GST activity N 11 IU/L GST activity N 13 IU/L
68.6 78.6
65.9 56.5
63.2 28.9
71.1 92.1
2.18 3.67
4.21 (1.45–12.45) 4.75 (1.07–24.04)
0.006 0.038
p N 0.05, non-significant differences (ns).
Table 3 Descriptive statistics for clinical and molecular variables in the enlarged affected group. Studied variables (Mean ± SD)
Age (years) Expanded CAG repeat number GST activity (IU/L) Age at onset (years) Disease duration (years) p N 0.05, non-significant differences (ns).
Affected patients
Student's t-test
Full data (n = 121)
Males (n = 65)
Females (n = 56)
t
p
41.4 40.9 12.5 28.2 13.2
40.8 40.9 12.92 27.4 13.42
42.1 40.9 11.92 29.1 13.0
−0.63 −0.01 1.86 −0.88 0.28
0.53(ns) 0.99(ns) 0.07(ns) 0.38(ns) 0.78(ns)
± ± ± ± ±
11.15 3.83 2.99 10.49 8.04
± ± ± ± ±
10.54 3.09 3.04 9.29 7.22
± ± ± ± ±
11.87 4.57 2.87 11.75 8.95
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D. Almaguer-Gotay et al. / Journal of the Neurological Sciences 341 (2014) 41–45
Fig. 2. Evidences for a non-significant association (p N 0.05) between glutathione-S-transferase activity and the age at disease onset. A) Glutathione-S-transferase activity by tercils of age at onset. An inverted-U distribution is noted. B) Glutathione-S-transferase activity by tercils of residuals of age at onset after adjustment by linear regression for the expanded CAG repeat number. An inverted-U distribution is noted.
Nonetheless, further studies including patients affected by additional neurodegenerative disorders as comparison groups will be needed to verify this observation. The presymptomatic group showing no significant increase in GST activity could be due to the fact that only 18.4% of them have a 50% risk or greater to become affected, so as a group they are closer to controls. Probably, the tendency observed for an increased GST activity with an advancing risk to become affected could turn out to be more evident in a larger cohort of presymptomatic individuals, and it suggests a presymptomatic condition as a multi-stage transition phase between a normal and a clinically relevant pathological physiology. Previous studies
supporting this notion were not based on biochemical findings but on the occurrence of saccadic velocity and sleep disturbances [16,17], and on evidences of pontocerebellar system atrophy [18]. The increase in the GST activity in both, affected and presymptomatic individuals, could be linked to the occurrence of oxidative stress. Actually, oxidative stress is present in affected SCA2 patients [4,5] and cellular models [19], and there is also a decreased antioxidant status in presymptomatic individuals, suggesting the existence of oxidative stress since the pre-clinical stages in SCA2 (unpublished data). This investigation relies on the use of serum samples for the measurement of GST activity. As in serum GST pi and alpha classes are the
Fig. 3. Regression of disease severity (SARA total score) and disease progression rate (SARA total score / disease duration) on clinical and molecular variables under study. Glutathione-Stransferase (GST) activity has little impact on the disease severity and the disease progression rate. The plots show data points from 50 SCA2 subjects of known SARA and disease progression rate scores. The straight lines represent the best-fit simple linear regression to the data. 95% CI regression bands are also shown. SARAts—SARA total score; (CAG)n—CAG repeat number at expanded ATXN2 alleles; AO—age at onset; DD—disease duration.
D. Almaguer-Gotay et al. / Journal of the Neurological Sciences 341 (2014) 41–45
ones with the highest levels in comparison to other members of the GST family [20], and 4-hydroxy-2-nonenal (4-HNE) – a major product of lipid peroxidation – is the main substrate for these two enzymes [21], the observed increase in GST activity could be reflecting an increase in lipid peroxidation and oxidative stress. In addition, it is well known that the expression or activation of some GST isoenzymes is induced by oxidative stress [22]. Particularly, incubation of H9c2 cells with 4-HNE at noncytotoxic concentrations, results in a significant increase in GST-A1 mRNA levels [23]. Furthermore, 4-HNE pre-treated cells exhibit increased resistance to injury elicited by subsequent cytotoxic concentrations of 4-HNE [23]. Also, GST pi activity in RL34 cells is induced by HNE in a time-dependent manner [24]. Similarly, it is known that reactive oxygen species (ROS) play a central role in regulating apoptosis. Particularly, 4-HNE has been involved in the activation of apoptosis via c-Jun N-terminal kinase (JNK) and caspase-3 mediated signaling [25,26]. Remarkably, GSTs are major factors regulating the intracellular concentrations of 4-HNE and they can influence the susceptibility to toxic effects, particularly when 4-HNE and GST levels are altered in disease states [27]. Although an association between oxidative stress and apoptosis has not yet been experimentally proven for SCA2, the increased GST activity in affected and presymptomatic individuals probably represents a cellular stress response to counteract the damaging effects of free radicals and to prevent apoptotic cell death of the targeted neuronal sub-populations. Moreover, it has been found that a significant increase of intracellular GST activity in striatal cells of a Huntington's disease knock-in model [28], and the expression of polyQ expanded ATXN7 in a PC12 model result in a concomitant increase in ROS levels and aggregation of the disease protein [29]. Also, the induction of mutant ATXN7 leads to a decrease in catalase levels and a progressive increased expression of GSTA-3 [29]. These observations suggest a relevant role for GSTs not only in SCA2 but in additional polyglutamine disorders. An increased GST activity in SCA2 is expected to have an impact on phenotypic markers and to be associated to the CAG repeat size. However the GST activity did not correlate with the CAG repeat expansion and failed to have any significant impact on disease severity or progression, suggesting that GSTs pi and alpha might not be key factors in the SCA2 neurodegenerative process. Hypothetically, the early and sustained expression of mutant ataxin-2 per se stimulates cellular free radical production that progressively enhances GST activity following non-linear dynamics and pathways not directly related to the primary pathogenic mechanisms. Alternatively, this lack of association could be caused by GST activity variability due to differential patients' exposure to xenobiotics, diet, toxic habits and chronic health conditions other than SCA2. In conclusion, these findings provide evidences for an in vivo alteration of GST activity in SCA2, supporting a role for free radical damage in the pathophysiological process. This study also shows the need to address the identification of reliable oxidative stress markers for SCA2 to be used in future therapeutic trials. Conflict of interest There is no conflict of interest. Acknowledgments We are deeply indebted to the patients and relatives for their valuable collaboration, to the Cuban Public Health and Science and Technology Ministries for their cooperation with this research. We are indebted to PhDs Luis Velázquez Pérez, Gilberto Sánchez Cruz, René Delgado, Julio Cesar García and MSc. Gretel Riberón for their guidance in the study of oxidative stress in Cuban SCA2 patients. We are also grateful to Dr. Patrick MacLeod for the technical support and to PhD Maritza Ulloa and MSc Pablo Bahr for their critical review of the paper, and to Prof. Gustavo Pérez Quintero for correcting the use of the English language.
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Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Role of glutathione S-transferases in the spinocerebellar ataxia type 2 clinical phenotype D. Almaguer-Gotay ⁎, L.E. Almaguer-Mederos, R. Aguilera-Rodríguez, A. Estupiñán-Rodríguez, Y. González-Zaldivar, D. Cuello-Almarales, J.M. Laffita-Mesa, Y. Vázquez-Mojena Center for the Research and Rehabilitation of Hereditary Ataxias—CIRAH, Cuba
a r t i c l e
i n f o
Article history: Received 11 September 2013 Received in revised form 21 March 2014 Accepted 24 March 2014 Available online 1 April 2014 Keywords: Oxidative stress Polyglutamine disease Spinocerebellar ataxia type 2 Glutathione S-transferase Antioxidant enzymes Presymptomatic
a b s t r a c t Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative and incurable hereditary disorder caused by a CAG repeat expansion mutation on ATXN2 gene. The identification of reliable biochemical markers of disease severity is of paramount significance for the development and assessment of clinical trials. In order to evaluate the potential use of glutathione-S-transferase (GST) activity as a biomarker for SCA2, a case–control study in 38 affected, presymptomatic individuals or healthy controls was conducted. An enlarged sample of 121 affected individuals was set to assess the impact of GST activity on SCA2 clinical expression. There was a significant increase in GST activity in affected individuals relative to controls, although sensibility and specificity were not high. GST activity was not significantly influenced by sex, age, disease duration or CAG repeat size and did not significantly influence disease severity markers. These findings show a disruption of in vivo GST activity in SCA2, suggesting a role for oxidative stress in the neurodegenerative process. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
2. Methods
Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative hereditary disorder caused by a CAG repeat expansion mutation on the first exon of ATXN2 gene. It has been widely characterized in its clinical, neurophysiological, molecular and epidemiological aspects [1,2]. However, further studies are needed for the identification of biochemical biomarkers of disease severity and progression. Several lines of evidence suggest glutathione S-transferase activity as a candidate biomarker in SCA2. Human glutathione S-transferases (GSTs, EC 2.5.1.18) are involved in the metabolism of xenobiotics, protection against oxidative stress and prevention of apoptosis [3]. Coincidentally, SCA2 has been linked to oxidative stress, particularly to altered activity of key antioxidant enzymes [4,5], and to increased apoptosis [6]. Therefore, it is expected that the GST activity being altered in SCA2 mutation carriers as a response to the neurodegenerative process is caused by the expression and dysfunction of mutant ataxin-2. The largest Cuban SCA2 population was studied in order to demonstrate the potential value of GST activity as a biomarker for SCA2. The association between serum GST activity and expanded CAG repeat size, age at onset, disease duration, disease severity score and disease progression rate was also assessed.
2.1. Subjects
⁎ Corresponding author at: Libertad No. 26. Entre 12 y 14. Rpto: El llano, Holguín CP: 80100, Cuba. Tel.: +53 24 462823. E-mail addresses: [email protected], [email protected] (D. Almaguer-Gotay).
http://dx.doi.org/10.1016/j.jns.2014.03.045 0022-510X/© 2014 Elsevier B.V. All rights reserved.
A case–control study was conducted to assess a possible association between GST activity and SCA2. Thirty-eight molecularly confirmed SCA2 affected individuals (11 men and 27 women), aged 17 to 62 years, were recruited at the Center for the Investigation and Rehabilitation of Hereditary Ataxias, Holguín, Cuba. Age and sex matched groups of presymptomatic individuals (aged 18 to 66 years) and healthy controls (aged 22 to 64 years) were included in the study. Affected and presymptomatic subjects were considered separately or as a single group (“mutation carriers”). The sample of affected individuals was enlarged to 121 subjects (aged 17 to 68 years) to evaluate the contribution of GST activity to the age at onset variability. Informed consent was obtained from the affected, presymptomatic and control individuals.
2.2. Clinical assessment A complete neurological examination was conducted and the clinical diagnosis of SCA2 was based on positive family history and disease manifestation such as ataxic gait, dysarthria, dysmetria, dysdiadochokinesia and slowing of saccadic eye movements. The age at onset was defined as the onset of motor impairment, and disease duration as the years from onset of clinical expression to the last examination. Disease severity was assessed using the Scale for the Assessment and Rating of Ataxia (the
42
D. Almaguer-Gotay et al. / Journal of the Neurological Sciences 341 (2014) 41–45
SARA) [7]. The disease progression rate at presentation was measured as the SARA score/disease duration.
3. Results 3.1. Association of GST activity with SCA2
2.3. CAG repeat size determination Genomic DNA was isolated from peripheral blood leukocytes using a standard protocol. The ATXN2 CAG repeat length was assessed by polymerase chain reaction (PCR) and the amplification with the previously published UH10 and UH13 oligonucleotide primers [8]. Aliquots of the PCR products were then sized by fragment analysis on an ALF Express II apparatus (Amersham Biosciences, Sweden) in comparison to internal and external size markers using Allele Links 1.0 software (Amersham Biosciences, Sweden). Cases with 32 or more repeats were designated as ATXN2 gene carriers in accordance with published association with disease expression [9].
2.4. GST activity measurement Serum was obtained by blood centrifugation at 3000 g for 5 min. It was frozen immediately after and stored at −20 °C until analyses. GST activity was measured by a spectrophotometric method as described by Habig [10]. Briefly, GST activity was determined using 25 mmol/L 1-chloro-2,4-dinitrobenzene (CDNB) and 20 mmol/L reduced glutathione (GSH) as cosubstrates in 0.5 mol/L potassium phosphate buffer (pH 6.5). The reaction was monitored at 340 nm using a BioMate spectrophotometer (ThermoSpectronic, USA).
In the full data of affected, presymptomatic and control individuals, GST activity varied between 6.77 and 18.75 IU/L, with a mean of 10.66 ± 2.39 IU/L. GST activity did not correlate significantly with the age (r = −0.072; p = 0.43). There was a tendency for females to have a higher GST activity (10.95 ± 2.40 IU/L) relative to males (10.02 ± 2.24 IU/L); however, this difference failed to reach statistical significance (t = −1.87; p = 0.064). The composition by sex, age and CAG repeat size of the studied groups is shown in Table 1. There were no significant differences according to age for these three groups; however, affected individuals showed significantly higher CAG repeat lengths than presymptomatic individuals (p b 0.001). Mutation carriers showed an increased GST activity in comparison to controls, which is due to a highly significant increase in GST activity in affected individuals (11.9 ± 2.24 IU/L). Actually, presymptomatics showed an increased GST activity (10.3 ± 2.11 IU/L) in comparison to the controls (9.77 ± 2.33 IU/L) but the difference was not significant (Fig. 1A and B). The age showed no significant correlation with GST activity in any studied group (p N 0.05). Control females showed a significantly higher GST activity relative to control males (p b 0.001); this finding was not replicated in affected or presymptomatic individuals (p N 0.05). As presymptomatic patients represent a transition state between healthy and affected individuals, the impact of increasing the risk to become affected on the GST activity was assessed. Although there was a tendency for GST activity to increase with the increase in the risk to become affected, no significant differences were found between individuals belonging to different risk classes (F = 0.017; p = 0.98) (Fig. 1C).
2.5. Statistical analysis Descriptive statistics were used to describe central tendencies and dispersion of the variables under study. A normal distribution for the variables was verified using the Shapiro–Wilk test. Comparison of GST activity between mutation carriers (clinically affected subjects plus presymptomatic individuals) and control individuals was performed by means of the Student's t-test. This test was also used for comparing GST activity between sexes. One-way ANOVA was used as a means of comparison in the circumstances when more than two groups were involved. Tercils were defined according to frequency distributions for the risk to become affected in presymptomatic individuals, age at onset and residual age at onset after adjustment by linear regression for the expanded CAG repeat number in affected subjects. The risk to become affected was calculated by survival analysis according to the CAG repeat number and current age for each presymptomatic individual as previously described [11]. Correlations between GST activity, age, age at onset, disease duration and expanded CAG repeat were assessed by Pearson's correlation test, once they were considered as parametric variables. Statistical significance was defined as p b 0.05. All statistics were computed in SPSS software (version 15.0) [12]. Positive predictive values, negative predictive values, sensitivity, specificity, likelihood ratios and odds ratio were calculated using EpiMax algorithm [13].
3.2. Sensitivity and specificity GST activity at different cut-off values was analyzed to determine its sensitivity and specificity for diagnosing SCA2 (Table 2). The odds ratio to be diagnosed with SCA2 for a GST activity value of 11 IU/L was 4.21 with a specificity of 71.1 and a sensitivity of 63.2. This would indicate that a false-positive rate for this potential diagnostic biomarker within the general population would be 31.4% (a positive predictive value of 68.6) and that 34.1% (a negative predictive value of 65.9%) of those individuals tested that are likely to have SCA2 would be falsely detected as normal. Predictive values were not better for a cut-off value of 13 IU/L. 3.3. Significance of GST activity for the SCA2 clinical phenotype In order to get insight into the significance of GST activity at the age of onset, an enlarged sample of 121 affected patients was studied. In this data, sex did not significantly influence on clinical or molecular SCA2 markers, nor on GST activity (Table 3). Also, GST activity was not significantly influenced by age, disease duration or CAG repeat number (p N 0.05), and had no influence on the age at onset (F = 1.095; p = 0.34), even after adjustment for the expanded CAG repeat number (F = 0.335; p = 0.72) (Fig. 2).
Table 1 Descriptive statistics for general variables included in the study.
Sex (male/female) Age (years) (Mean ± SD) Expanded CAG repeat number (Mean ± SD) Non-significant differences (ns). a One-way ANOVA. b Student's t-test; p N 0.05.
Affected (n = 38)
Presymptomatics (n = 38)
Controls (n = 38)
p
11/27 38.7 ± 10.09 42.7 ± 4.73
11/27 37.7 ± 10.49 36.8 ± 2.26
11/27 37.4 ± 9.77 –
– 0.86a(ns) b0.001b
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43
Fig. 1. Glutathione-S-transferase activity by clinical status. A) Comparison of GST activity between mutation carriers (affected + presymptomatics) and healthy subjects. A significant increase in GST activity was noted for mutation carriers related to the controls (**p = 0.005). B) Comparison of GST activity between affected, presymptomatic and healthy subjects. A significant increase in GST activity was noted for affected individuals related to presymptomatic and control individuals (***p b 0.001; **p = 0.005). C) GST activity in presymptomatic individuals according to the risk of becoming affected. A non-significant increase in GST activity as the individual's risk advances was obtained (p N 0.05).
4. Discussion
In a subsample of 50 affected individuals it was possible to obtain the SARA and disease progression rate scores. The SARA total score was not significantly influenced by sex or age (p N 0.05), but it showed significant associations with age at onset, disease duration and CAG repeat number. In addition, GST activity had a statistically significant but low impact on the SARA total score (Fig. 3). At multiple linear regression only disease duration and the CAG repeat number were significant predictors of the SARA total score (R2 = 0.45). Similarly, disease progression rates were not significantly influenced by sex (p N 0.05) but it showed significant associations with age at onset, CAG repeat number and GST activity (Fig. 3). However, at multiple linear regression only age at onset and the CAG repeat number were predictors of disease progression rates (R2 = 0.74).
Human glutathione S-transferases are widely expressed and provide the most important line of cellular defense against electrophilic genotoxic endogenous compounds and xenobiotic chemicals. They are also involved in the protection against oxidative stress and prevention of apoptosis [3, 14,15]. The present study showed that increased GST activity is associated to SCA2, probably even since the presymptomatic stage. In fact, the affected and presymptomatic individuals showed 21.8% and 5.5% increases in GST activity, respectively, relative to age and sex matched controls. However, GST activity did not show high sensitivity and specificity; therefore, its utility for diagnostic purposes is very limited.
Table 2 Positive predictive value (PPV), negative predictive value (NPV), sensitivity, specificity, likelihood ratio (LR), odds ratio (OR) and p-values for GST activity. Affected vs. controls
PPV (%)
NPV (%)
Sensitivity (%)
Specificity (%)
LR
OR (95% CI)
p
GST activity N 11 IU/L GST activity N 13 IU/L
68.6 78.6
65.9 56.5
63.2 28.9
71.1 92.1
2.18 3.67
4.21 (1.45–12.45) 4.75 (1.07–24.04)
0.006 0.038
p N 0.05, non-significant differences (ns).
Table 3 Descriptive statistics for clinical and molecular variables in the enlarged affected group. Studied variables (Mean ± SD)
Age (years) Expanded CAG repeat number GST activity (IU/L) Age at onset (years) Disease duration (years) p N 0.05, non-significant differences (ns).
Affected patients
Student's t-test
Full data (n = 121)
Males (n = 65)
Females (n = 56)
t
p
41.4 40.9 12.5 28.2 13.2
40.8 40.9 12.92 27.4 13.42
42.1 40.9 11.92 29.1 13.0
−0.63 −0.01 1.86 −0.88 0.28
0.53(ns) 0.99(ns) 0.07(ns) 0.38(ns) 0.78(ns)
± ± ± ± ±
11.15 3.83 2.99 10.49 8.04
± ± ± ± ±
10.54 3.09 3.04 9.29 7.22
± ± ± ± ±
11.87 4.57 2.87 11.75 8.95
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Fig. 2. Evidences for a non-significant association (p N 0.05) between glutathione-S-transferase activity and the age at disease onset. A) Glutathione-S-transferase activity by tercils of age at onset. An inverted-U distribution is noted. B) Glutathione-S-transferase activity by tercils of residuals of age at onset after adjustment by linear regression for the expanded CAG repeat number. An inverted-U distribution is noted.
Nonetheless, further studies including patients affected by additional neurodegenerative disorders as comparison groups will be needed to verify this observation. The presymptomatic group showing no significant increase in GST activity could be due to the fact that only 18.4% of them have a 50% risk or greater to become affected, so as a group they are closer to controls. Probably, the tendency observed for an increased GST activity with an advancing risk to become affected could turn out to be more evident in a larger cohort of presymptomatic individuals, and it suggests a presymptomatic condition as a multi-stage transition phase between a normal and a clinically relevant pathological physiology. Previous studies
supporting this notion were not based on biochemical findings but on the occurrence of saccadic velocity and sleep disturbances [16,17], and on evidences of pontocerebellar system atrophy [18]. The increase in the GST activity in both, affected and presymptomatic individuals, could be linked to the occurrence of oxidative stress. Actually, oxidative stress is present in affected SCA2 patients [4,5] and cellular models [19], and there is also a decreased antioxidant status in presymptomatic individuals, suggesting the existence of oxidative stress since the pre-clinical stages in SCA2 (unpublished data). This investigation relies on the use of serum samples for the measurement of GST activity. As in serum GST pi and alpha classes are the
Fig. 3. Regression of disease severity (SARA total score) and disease progression rate (SARA total score / disease duration) on clinical and molecular variables under study. Glutathione-Stransferase (GST) activity has little impact on the disease severity and the disease progression rate. The plots show data points from 50 SCA2 subjects of known SARA and disease progression rate scores. The straight lines represent the best-fit simple linear regression to the data. 95% CI regression bands are also shown. SARAts—SARA total score; (CAG)n—CAG repeat number at expanded ATXN2 alleles; AO—age at onset; DD—disease duration.
D. Almaguer-Gotay et al. / Journal of the Neurological Sciences 341 (2014) 41–45
ones with the highest levels in comparison to other members of the GST family [20], and 4-hydroxy-2-nonenal (4-HNE) – a major product of lipid peroxidation – is the main substrate for these two enzymes [21], the observed increase in GST activity could be reflecting an increase in lipid peroxidation and oxidative stress. In addition, it is well known that the expression or activation of some GST isoenzymes is induced by oxidative stress [22]. Particularly, incubation of H9c2 cells with 4-HNE at noncytotoxic concentrations, results in a significant increase in GST-A1 mRNA levels [23]. Furthermore, 4-HNE pre-treated cells exhibit increased resistance to injury elicited by subsequent cytotoxic concentrations of 4-HNE [23]. Also, GST pi activity in RL34 cells is induced by HNE in a time-dependent manner [24]. Similarly, it is known that reactive oxygen species (ROS) play a central role in regulating apoptosis. Particularly, 4-HNE has been involved in the activation of apoptosis via c-Jun N-terminal kinase (JNK) and caspase-3 mediated signaling [25,26]. Remarkably, GSTs are major factors regulating the intracellular concentrations of 4-HNE and they can influence the susceptibility to toxic effects, particularly when 4-HNE and GST levels are altered in disease states [27]. Although an association between oxidative stress and apoptosis has not yet been experimentally proven for SCA2, the increased GST activity in affected and presymptomatic individuals probably represents a cellular stress response to counteract the damaging effects of free radicals and to prevent apoptotic cell death of the targeted neuronal sub-populations. Moreover, it has been found that a significant increase of intracellular GST activity in striatal cells of a Huntington's disease knock-in model [28], and the expression of polyQ expanded ATXN7 in a PC12 model result in a concomitant increase in ROS levels and aggregation of the disease protein [29]. Also, the induction of mutant ATXN7 leads to a decrease in catalase levels and a progressive increased expression of GSTA-3 [29]. These observations suggest a relevant role for GSTs not only in SCA2 but in additional polyglutamine disorders. An increased GST activity in SCA2 is expected to have an impact on phenotypic markers and to be associated to the CAG repeat size. However the GST activity did not correlate with the CAG repeat expansion and failed to have any significant impact on disease severity or progression, suggesting that GSTs pi and alpha might not be key factors in the SCA2 neurodegenerative process. Hypothetically, the early and sustained expression of mutant ataxin-2 per se stimulates cellular free radical production that progressively enhances GST activity following non-linear dynamics and pathways not directly related to the primary pathogenic mechanisms. Alternatively, this lack of association could be caused by GST activity variability due to differential patients' exposure to xenobiotics, diet, toxic habits and chronic health conditions other than SCA2. In conclusion, these findings provide evidences for an in vivo alteration of GST activity in SCA2, supporting a role for free radical damage in the pathophysiological process. This study also shows the need to address the identification of reliable oxidative stress markers for SCA2 to be used in future therapeutic trials. Conflict of interest There is no conflict of interest. Acknowledgments We are deeply indebted to the patients and relatives for their valuable collaboration, to the Cuban Public Health and Science and Technology Ministries for their cooperation with this research. We are indebted to PhDs Luis Velázquez Pérez, Gilberto Sánchez Cruz, René Delgado, Julio Cesar García and MSc. Gretel Riberón for their guidance in the study of oxidative stress in Cuban SCA2 patients. We are also grateful to Dr. Patrick MacLeod for the technical support and to PhD Maritza Ulloa and MSc Pablo Bahr for their critical review of the paper, and to Prof. Gustavo Pérez Quintero for correcting the use of the English language.
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