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The Influence of ADORA2A on Levodopa-Induced Dyskinesia
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.344646
Keywords: ADORA2A, genetics, levodopa, levodopa-induced dyskinesia, motor complications, neurogenetics, Parkinson's disease, polymorphism
Parkinson's Disease (PD) occurs by the deterioration of dopamine-related nigrostriatal neurons, and on treatment, levodopa is the gold standard. Even though it is the optimal drug, its unwanted side effect is hyperkinetic movements as levodopa-induced dyskinesias (LID).[1],[2],[3],[4],[5],[6] Dopaminergic transmission genes are the most accountable factors in the pathophysiology that lead to this side effect.[3],[7] Another genetic development that has recently featured is on the adenosine pathway. Adenosine and dopamine D2 receptors are localized together; this association leads adenosine to act as an agonist through the A2A receptors (A2ARs), which are encoded by the ADORA2A gene.[8],[9] A2AR antagonists reduce the functions of dopamine, and D2 receptor agonists, therefore, prevent the emergence of LID.[7],[8],[10],[11],[12] According to the implications, we set out to define the relationships of ADORA2A polymorphisms (rs2298383, rs3506042, and rs5751876) on Turkish-sampled PD patients.
Subjects We evaluated 172 patients diagnosed as PD between the dates of September 2017 and September 2018 at the Movement Disorders outpatient clinic in the University of Health Sciences, Erenkoy Mental Health and Neurological Disorders Research and Training Hospital in Istanbul, Turkey. We separated the patients into two groups as the patient group (67 PD patients with LID) and the control group (105 PD patients without LID) [Figure 1]. Inclusion criteria were defined as follows: A diagnosis of PD considering as the Movement Disorder Society Clinical Diagnostic Criteria for PD,[13] using levodopa at least for 1 year, to be between the age of 30 and 85, and to have no history of secondary Parkinsonism More Details. We excluded the patients who did not give informed consent or take levodopa for medication. We collected the demographical data by patient evaluations. Patients were examined by using the Turkish version of Unified Parkinson's Disease Rating Scale (UPDRS)[14] and the Hoehn–Yahr Rating Scale (HYRS).[15] We determined to diagnose LID in the on phase of levodopa intake on examination by two different neurologists. The study received approval from the hospital's ethical board, and we provided individuals' written informed consent.
DNA extraction Primer sequences of polymorphisms for PCR amplification were shown on [Table 1]. We protected the peripheral blood samples in ethylenediaminetetraacetic acid at 4°C until the use. Dodecyl-trimethyl-ammonium-bromide/cetyl-trimethyl-ammonium-bromide method was used to isolate the DNAs. DNA concentrations and purification were verified by spectrophotometry device (NanoPhotometer, Implen, GmbH, Germany). The samples were stored at −20°C until the further procedure. Single nucleotide polymorphisms (SNPs) associated with ADORA2A were identified and characterized using real-time PCR (rt-PCR) (LightCycler 480 rt-PCR System, Roche Diagnostics, Mannheim, Germany) and HRM analysis. The PCR mixture (20 μL total volume) contained 2 μL of DNA (50 ng), 10 μL of Master Mix (Fast Plus Eva Green qPCR Master Mix-Low Rox from Biotium, Inc.), 1 μL of SNP primer mix (25 mM), and 7 μL of dH2O. We performed the amplification in the following order: Preincubation for 2 min at 95°C and 40 cycles, consisting at 95°C and 10 s for denaturation, 60°C and 30 s for annealing, and 72°C and 30 s for extending. After the amplification, an HRM curve was generated according to as follows: Denaturation for 1 min at 95°C, annealing for 1 min at 40°C, elongation 60°C for 1 min, and cooling at 40°C for 10 s. Analyzation was achieved by the HRM program while monitoring the change in fluorescence signal in real time. A homozygous DNA sample was compared to heterozygous DNA specimen shaping as heteroduplexes, ending in a different shape of the melting bow. In contrast, diverse genotypes of homozygous DNA specimen were distinguished by a melting temperature (Tm) shift rather than an altered bow form.
Statistical analysis We evaluated the categorical variables by Chi-squared and Fisher–Freeman–Halton tests. Quantitative variables were measured by the Student's t-test with normal distribution, and Kruskal Wallis and Mann–Whitney U-tests were assessed without normal distribution. Multivariate analysis included rs35060421 polymorphism, sex, disease duration, age of onset, the daily amount of levodopa, and duration of levodopa therapy. Allele frequencies were assessed by gene reckoning. P < 0.05 was considered significant. We applied logistic regression analyses, with robust standard faults, to determine if LID is affected by the rs35060421 polymorphism independently after eliminating dependent variables as sex, length of the illness, age at onset, the daily amount of levodopa, and length of levodopa therapy. We executed the statistical analysis using the Number Cruncher Statistical System 2007 (Kaysville, Utah, USA).
There was a statistically significant difference in clinical characteristics and demographical data of the patients on age at onset, length of the disease, daily intake of levodopa, the length of levodopa medication, the UPDRS, and HYRS scores as shown on [Table 2]. The patient group exhibited more prolonged duration levodopa therapy, took higher doses of medication daily, had a younger age at onset, and had greater HYRS and UPDRS scores. In the patient group, motor fluctuations were also found more recurrent significantly (P = 0.001). Analysis of genotypes and polymorphism frequencies was carried out for the C/T change in intron 1 of rs2298383, and exon changes C/T in rs5751876 and C/T in rs35060421. We did not find any significant differences amid the groups on neither genotypes nor allele frequencies for all three polymorphisms [Table 3].
Although there were no statistical differences in allele frequencies, the rs35060421 polymorphism took our attention because we observed that this polymorphism repeated more frequently in both groups. According to this observation, even though we did not find an association statistically, we decided to perform a logistic regression analysis to determine the occurrence of the rs35060421 polymorphism if it would have been a dependent variable to link for developing LID but found no relationship (P = 0.263). In the light of these findings, we observed a significant association between LID and the age at onset, female sexuality, LD daily dosage, and duration of LD therapy (P = 0.001, P = 0.0035, P = 0.036, P < 0.05, respectively). The duration of disease and age at onset were determined as significant according to univariate analysis on statistics; however, no significant differences were noticed on multivariate analysis. Thus, we concluded that the rs35060421 polymorphism might be a risk factor for developing LID independently. On univariate analysis, the odds ratio of rs35060421 polymorphism was 1.39, but without the involvement of dependent variables, the odds ratio increased to 1.608 on multivariate analysis. This increase suggests that the allele might be either an independent risk factor to develop dyskinesia or the risk may increase with other parameters' contributions.
It has been presumed that A2AR antagonists may enhance the effects of dopaminergic treatments. Considering this, we studied specific polymorphisms of the ADORA2A gene (rs5751876, rs2298383, rs35060421) and try to understand their possible relationships with LID development.[8],[9],[11],[16] Although the results showed us that higher sample number might produce different results as a possible, current result in the Turkish sample indicated that rs2298383 and rs5751876 alleles in the ADORA2A gene in PD patients might not be related to LID. This outcome shows that there could be another drug-metabolizing enzymes, drug receptors, or other reasons that might cause LID. Therefore, this study's result is meaningful in terms of rs2298383 and rs5751876 alleles in the ADORA2A gene and may not cause LID in terms of Turkish sample. For that, it can be suggested that A2AR should be investigated to cause such an effect in patients on varied populations. Ivanova et al.[17],[18] studied the rs35060421 allele if related to the dyskinesia in hyperkinetic movement disorders, but found no association. In our study, we observed that the frequency of this polymorphism was higher in both groups, but when we analyze with statistical methods to support our observation, we did not find a significant relationship between the allele and LID. Even though the result was insignificant, we performed the multivariate analysis, and it led us to conclude that this allele might reveal itself as an independent risk factor in studies with a sufficient number of patients. The rs5751876 allele was chosen because of its reducing effect on the affinition of A2AR for adenosine, thus enhancing dopamine transmission.[19],[20],[21] A study investigating the relationship between this variant on PD patients revealed no substantial difference.[22] Similarly, we intended to analyze this polymorphism on Turkish-sampled PD patients but could not find an association. Another allele that captivated our minds was the rs2298383 polymorphism. It affects the transcription of the ADORA2A gene and leads to reduce the protein expression.[19],[22] Greenbaum et al.[23] found an increased risk between this allele and LID, in earlier stages of the disease. In two large studies, this allele was found to be associated with increasing LID development and suggesting that it might be a risk factor.[12],[22] According to relevant literature, we investigated rs2298383 but found no association. A concern about this study might be the differences in the length of illness that did not match in both groups. One of the opinions reported on this matter is motor fluctuations and dyskinesia occur as the same frequency and severity regardless of whether levodopa was given in early stages or late. Experimental and patient-based studies support this opinion.[24],[25],[26],[27] Comparing the groups among the length of illness would not matter due to inequality; therefore, it did not prevent us from determining the exact influence of the polymorphisms. Consequently, we did not find any effect of these alleles on LID development statistically. Since there is no significant difference in polymorphisms between the groups, it has been revealed that the affecting factor caused LID is the severity and daily levodopa dosage rather than ADORA2A polymorphisms. Therefore, our results are consistent with previous studies.[24] According to this observation, our study is the first that compares these three polymorphisms if they influence LID independently in terms of the Turkish sample. This result shows us that LID occurs by the severity and total amount of levodopa daily, but not the ADORA2A polymorphisms. Following that, it can be said that these three polymorphisms may not be related to LID. As a secondary result of our study, we tried to understand whether dyskinesia's development is related to the severity of the disease and the daily amount of levodopa. Therefore, it did not prevent us from including the early-staged patients to the control group and compare them with the patient group; consequently, we found a significant difference. Thus, as a secondary result of our study, we presented a statistical significance of the disease's severity and a higher amount of levodopa daily. This result supports the fallouts in the relevant literature.[27] Although the major limitation of our study was an insufficient amount of patients, this study is the first to investigate these polymorphisms in Turkish PD patients and suggests that the results should be considered as positive exploratory findings. The implication of this study was to specify the impact of ADORA2A gene polymorphisms regarding the Turkish sample. There have been no studies of rs35060421 polymorphism in PD patients worldwide, and supplementary investigations with greater-numbered study sizes are required to determine its influence. There are many gaps in our knowledge surrounding the ADORA2A gene, which would benefit from further research, including realistic evaluations to extend and further test different polymorphisms. In conclusion, as we learn more about LID's genetics, it is becoming abundantly clear that the polymorphisms of the ADORA2A gene might play an essential part in treatment. However, there may be more explainable factors about the ADORA2A gene polymorphisms. With detailed investigations and determinations on the specific polymorphisms, the treatment could be personalized, and patients might potentially experience a better quality of life without dyskinesia. Therefore, future studies on specific features of the ADORA2A gene's rs781339364, rs781051901, and rs774685876 polymorphisms are needed in terms of different populations. With this study, it can be said that the near-future road map points out new studies conduct more than the present. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Acknowledgements This study could not have been feasible without the generous support of fellows of the Yeditepe University Medical Faculty Genetics Department, Istanbul, Turkey. The authors are grateful for funding from the University of Health Sciences Scientific Research Project Unit. Financial support and sponsorship “University of Health Sciences Scientific Research Project Unit” supported this research with the project number of 2017/023, the date of approval was 03.07.2017. Conflicts of interest There are no conflicts of interest.
[Figure 1]
[Table 1], [Table 2], [Table 3]
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