Motor Fluctuations Development Is Associated with Non-Motor Symptoms Burden Progression in Parkinson’s Disease Patients: A 2-Year Follow-Up Study

Objective: The aim of the present study was to analyze the progression of non-motor symptoms (NMS) burden in Parkinson’s disease (PD) patients regarding the development of motor fluctuations (MF). Methods: PD patients without MF at baseline, who were recruited from January 2016 to November 2017 (V0) and evaluated again at a 2-year follow-up (V2) from 35 centers of Spain from the COPPADIS cohort, were included in this analysis. MF development at V2 was defined as a score ≥ 1 in the item-39 of the UPDRS-Part IV, whereas NMS burden was defined according to the Non-motor Symptoms Scale (NMSS) total score. Results: Three hundred and thirty PD patients (62.67 ± 8.7 years old; 58.8% males) were included. From V0 to V2, 27.6% of the patients developed MF. The mean NMSS total score at baseline was higher in those patients who developed MF after the 2-year follow-up (46.34 ± 36.48 vs. 34.3 ± 29.07; p = 0.001). A greater increase in the NMSS total score from V0 to V2 was observed in patients who developed MF (+16.07 ± 37.37) compared to those who did not develop MF (+6.2 ± 25.8) (p = 0.021). Development of MF after a 2-year follow-up was associated with an increase in the NMSS total score (β = 0.128; p = 0.046) after adjustment to age, gender, years from symptoms onset, levodopa equivalent daily dose (LEDD) and the NMSS total score at baseline, and the change in LEDD from V0 to V2. Conclusions: In PD patients, the development of MF is associated with a greater increase in the NMS burden after a 2-year follow-up.


Introduction
Parkinson's disease (PD) is a progressive neurodegenerative disorder causing motor and non-motor symptoms (NMS) that result in disability, loss of patient autonomy, and diminished quality of life (QoL) [1]. From a pathophysiological point of view, motor symptoms in PD are attributed to the degeneration of the dopaminergic nigrostriatal system [2]. Nevertheless, increasing evidence has shown that PD is a multisystem disorder characterized also by the degeneration of the mesocortical dopaminergic system, the noradrenergic system of the locus coeruleus, the serotonergic system of the dorsal raphe nuclei, and the cholinergic system of the nucleus basalis of Meynert, as well as the histaminergic, peptidergic, and olfactory-related systems [3]. This explains the complexity in management of NMS in PD and why many therapeutic strategies are based on correcting the deficit of neurotransmitters other than dopamine [4]. However, NMS can be related to dopamine as well. Increasing dopamine activity not only in the striatum but also in other areas of the brain could improve some NMS such as attention, executive functions, apathy, depression, anxiety, restless legs and periodic limb movements, urinary urgency, nocturia, dribbling of saliva, constipation, pain, or fatigue [5][6][7][8][9]. Moreover, NMS can be related to dopamine changes in brain and blood [10]. Thus, some patients can suffer from non-motor fluctuations (NMF) (i.e., NMS that fluctuate during the day) [11] or can experience motor fluctuations (MF) with the development of NMS during the OFF episodes (e.g., pain associated with dystonia) [12]. The close connection of NMF and MF strongly suggests that the strategies used to treat motor complications-namely, continuous dopaminergic stimulation-also apply for the therapy of NMF. Thus, a dopaminergic treatment reducing the daily OFF time can improve some NMS [9,13,14] or even the global NMS burden [15,16]. In line with this, we demonstrated recently in a cross-sectional study conducted in Spain that MF are frequent and associated with a greater NMS burden even during the first 5 years of disease duration [17]. This is of great importance because NMS burden is associated with a worse QoL and is also an independent predictor of clinically significant QoL impairment in PD [18,19].
In this context, we hypothesized that PD patients who develop MF in the short-term will increase their NMS burden compared with those patients who do not. Understanding this potential association is of interest because, in clinical practice, to detect MF is an essential point for the application of management strategies in PD [20]. The aim of the present study was to analyze the progression of NMS burden in PD patients from a Spanish cohort regarding the development of MF after a 2-year follow-up. Moreover, the change in health-related quality of life (HR-QoL) and global QoL (GQoL) was analyzed as well.

Material and Methods
PD patients without MF at baseline, who were recruited from 35 centers of Spain from the COPPADIS cohort [21] from January 2016 to November 2017 and evaluated again at 2-year follow-up, were included in the study. Methodology about COPPADIS-2015 study can be consulted in https://bmcneurol.biomedcentral.com/articles/10.1186/s12883-016-0548-9 accessed on 25 February 2016 [22]. This is a multicenter, observational, longitudinalprospective, 5-year follow-up study designed to analyze disease progression in a Spanish population of PD patients. All patients included were diagnosed according to UK PD Brain Bank criteria [22].
In PD subjects, information on sociodemographic aspects, factors related to PD, comorbidity, and treatment was collected at baseline (visit V0) and at 2 years ± 1 month (visit V2).  [22]. In all the scales/questionnaires, a higher score indicates a more severe affectation except for PD-CRS, PDSS, ADLS, and EUROHIS-QOL8, where it is opposite.
MF were defined according to the Unified Parkinson's Disease Rating Scale-Part IV (UPDRS-IV) [23]. Patients with a score = 0 on item-39 of the UPDRS-IV (UPDRS-IV-39) were considered as without MF whereas those with a UPDRS-IV-39 score ≥ 1 were defined as with MF. For this study, patients from the COPPADIS cohort who presented with MF (i.e., UPDRS-IV-39 ≥ 1) at baseline were excluded. In patients with MF, the motor assessment was made during the OFF state (without medication in the last 12 h) and during the ON state. On the other hand, the assessment was only performed without medication in patients without MF. Other data about motor complications were obtained from the UPDRS-IV.

Data Analysis
Data were processed using SPSS 20.0 for Windows. Only PD patients from the COP-PADIS cohort with data of the UPDRS-IV and NMSS total score collected at both visits, V0 and V2, were included in the analysis. For comparisons between patients with vs. without MF at V2, the Student's t-test, Mann-Whitney U test, Chi-square test, or Fisher test were used as appropriate (distribution for variables was verified by one-sample Kolmogorov-Smirnov test). Spearman's or Pearson's correlation coefficient, as appropriate, were used for analyzing the relationship between the change from V0 to V2 in continuous variables (NMSS, PDQ-39SI, EUROHIS-QOL8). Correlations were considered weak for coefficient values ≤ 0.29, moderate for values between 0.30 and 0.59, and strong for values ≥0.60. Marginal homogeneity tests were applied for comparing the frequency distribution of groups (NMS burden; from mild to very severe) between V0 and V2.
General linear model (GLM) repeated measure was used to test whether the mean differences of the total score and each domain of the NMSS, PDQ-39SI, and EUROHIS-QOL8 between the two visits (V0 and V2) were significant. The Bonferroni method was used as a post-hoc test after ANOVA. Cohen's d formula was applied for measuring the effect size; it was considered as follows: <0.2-Negligible; 0.2-0.49-Small; 0.50-0.79-Moderate; ≥0.80-Large. Age, gender, years from symptoms onset, H&Y stage, levodopa equivalent daily dose (LEDD) and the NMSS total score at baseline, and the change in LEDD from V0 to V2 were included as covariates in the model. The total score of each scale at V0 (NMSS, PDQ-39SI, and EUROHIS-QOL8) was included as covariate for the analysis of their domains.
With the aim to investigate if the development of MF from V0 to V2 was an independent factor associated with an increase in the NMS burden and impairment in the QoL, linear regression models with the change from V0 to V2 in the total score of the NMSS, PDQ-39SI, and EUROHIS-QOL8 (these variables as dependent variable in each model) were conducted. In all cases, the analysis was adjusted to age, gender, years from symptoms onset, H&Y stage, LEDD and the NMSS total score at baseline, and the change in LEDD from V0 to V2. The p-value was considered significant when it was <0.05.

Standard Protocol Approvals, Registrations, and Patient Consents
For this study, we received approval from the Comité de Ética de la Investigación Clínica de Galicia in Spain (2014/534; 02/DEC/2014). Written informed consent was obtained from all participants in this study. COPPADIS-2015 was classified by the AEMPS (Agencia Española del Medicamento y Productos Sanitarios) as a Postauthorization Prospective Follow-up study with the code COH-PAK-2014-01.

Data Availability
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.  Figure 1). At V0, the frequency of severe and very severe NMS burden was higher in those patients who developed MF at V2 compared with those who did not (27.5% vs. 18% and 18.7% vs. 12.1%, respectively; p = 0.011) ( Figure 2). (A) (B) Figure 1. (A) NMSS total score (y-axis) at baseline (V0) and after a 2-year follow-up (V2) in PD patients who developed MF at V2 (MF at V2 (PD-MFV2); N = 91) and those patients who did not develop MF at V2 (nonMF at V2 (PD-nonMFV2); N = 239). NMSS total score at V0, PD-MFV2 vs. PD-nonMFV2, p = 0.001; NMSS total score at V2, PD-MFV2 vs. PD-nonMFV2, p < 0.0001; change in the NMSS total score from V0 to V2 in PD-MFV2, p < 0.0001; change in the NMSS total score from V0 to V2 in PD-nonMFV2, p < 0.0001; comparison between the change in the NMSS total score from V0 to ; N = 91) and those patients who did not develop MF at V2 (nonMF at V2 (PD-nonMF V2 ); N = 239). NMSS total score at V0, PD-MF V2 vs. PD-nonMF V2 , p = 0.001; NMSS total score at V2, PD-MF V2 vs. PD-nonMF V2 , p < 0.0001; change in the NMSS total score from V0 to V2 in PD-MF V2 , p < 0.0001; change in the NMSS total score from V0 to V2 in PD-nonMF V2 , p < 0.0001; comparison between the change in the NMSS total score from V0 to V2 in PD-MF V2 vs. PD-nonMF V2 , p = 0.021. Data are presented as box plots, with the box representing the median and the two middle quartiles (25-75%). (B) Mean score on each domain of the NMSS at V0 and at V2 in both groups, PD-MF V2 and PD-nonMF V2 . At V0, the difference was significant between both groups in NMSS-1 (Cardiovascular) (p = 0.001), NMSS-2 (Sleep/fatigue) (p = 0.001), NMSS-4 (Perceptual symptoms) (p < 0.0001), and NMSS-9 (Miscellaneous) (p = 0.005). At V2, the difference was significant between both groups in all domains (p values from 0.024 to <0.0001) except in NMSS-5 (Attention/memory) (p = 0.364). p values were computed using the Kolmogorov-Smirnov, Mann-Whitney, and Wilcoxon tests. Mild outliers (O) are data points that are more extreme than Q1 − 1.5 * IQR or Q3 + 1.5 * IQR.
A greater increase in the NMSS total score from V0 to V2 was observed in those patients who developed MF at V2 (+16.07 ± 37.37) compared with those who did not develop MF (+6.2 ± 25.8) (p = 0.021) ( Table 1 and Figure 1). Two-hundred and two out of 330 patients (64.2%) presented at V2 a NMSS total score higher than at V0, but no differences between patients who developed MF vs. those who did not develop MF after the 2-year follow-up were observed (68.1% vs. 62.1%; p = 0.218). However, after the 2-year followup, the frequency of severe and very severe NMS burden was significantly higher in the group who developed MF (p < 0.0001) ( Figure 2). Applying GLM repeated measure and after adjustment to covariates (age, gender, years from symptoms onset, H&Y stage, LEDD and the NMSS total score at baseline, and the change in LEDD from V0 to V2), a significantly greater increase (34.6% vs. 17.9%; p = 0.005) in the NMSS total score was observed in patients who developed MF at V2 (from 46.34 ± 36.48 to 62.37 ± 44.15; Cohen's effect size = 0.57; p = 0.003) compared with those who did not develop MF (from 34.3 ± 29.07 vs. 40.5 ± 35.4; Cohen's effect size = 0.33; p < 0.0001) ( Table 2). An increase in the score of different domains from V0 to V2 was significant in both groups, with and without MF at V2, but there were no significant differences between them (Table 2 and Figure 1). Regarding QoL, the increase in the PDQ-39SI and decrease in EUROHIS-QOL8 total score ; N = 91) and those who did not developed MF at V2 (nonMF at V2 (PD-nonMF V2 ); N = 239). PD-nonMF V2 vs. PD-MF V2 at V0, p = 0.011; PD-nonMF V2 vs. PD-MF V2 at V2, p < 0.0001; change in PD-nonMF V2 from V0 to V2, p = 0.003; change in PD-MF V2 from V0 to V2, p = 0.001. p values were computed using the Chi-square and marginal homogeneity test.
A greater increase in the NMSS total score from V0 to V2 was observed in those patients who developed MF at V2 (+16.07 ± 37.37) compared with those who did not develop MF (+6.2 ± 25.8) (p = 0.021) ( Table 1 and Figure 1). Two-hundred and two out of 330 patients (64.2%) presented at V2 a NMSS total score higher than at V0, but no differences between patients who developed MF vs. those who did not develop MF after the 2-year follow-up were observed (68.1% vs. 62.1%; p = 0.218). However, after the 2-year follow-up, the frequency of severe and very severe NMS burden was significantly higher in the group who developed MF (p < 0.0001) (Figure 2). Applying GLM repeated measure and after adjustment to covariates (age, gender, years from symptoms onset, H&Y stage, LEDD and the NMSS total score at baseline, and the change in LEDD from V0 to V2), a significantly greater increase (34.6% vs. 17.9%; p = 0.005) in the NMSS total score was observed in patients who developed MF at V2 (from 46.34 ± 36.48 to 62.37 ± 44.15; Cohen's effect size = 0.57; p = 0.003) compared with those who did not develop MF (from 34.3 ± 29.07 vs. 40.5 ± 35.4; Cohen's effect size = 0.33; p < 0.0001) ( Table 2). An increase in the score of different domains from V0 to V2 was significant in both groups, with and without MF at V2, but there were no significant differences between them (Table 2 and Figure 1). Regarding QoL, the increase in the PDQ-39SI and decrease in EUROHIS-QOL8 total score indicating a QoL impairment between both visits, V0 and V2, was significantly greater in the group of patients who developed MF (PDQ-39SI, +35% vs. +26.5% (p = 0.002); EUROHIS-QOL8, −29.9% vs. −0.7% (p = 0.030)) ( Table 2). By domain and after adjustment to covariates including the PSQ-39SI score at V0, the increase on the score of "pain and discomfort" domain in the group who developed MF at V2 (from 28.55 ± 20.01 to 32.87 ± 24.33; Cohen's effect size = 0.30; p = 0.015) was significantly higher (p = 0.039) compared with patients who did not develop MF (from 20.65 ± 18.82 to 23.97 ± 22.12; Cohen's d effect size = 0.16; p = 0.071) ( Table 2). The mean score on all domains of the PDQ-39SI was the highest in patients who developed MF after the 2-year follow-up at V2 and the lowest in patients who did not develop MF, at V0 (Figure 3). A moderate correlation was observed between the change from V0 to V2 in the NMSS total score and the change in the PDQ-39SI in the whole cohort (N = 320; r = 0.402; p < 0.0001) and in both groups, patients with (N = 91; r = 0.328; p = 0.002) and without MF (N = 239; r = 0.433; <0.0001) at V2. However, the correlation between the change in the total score of the NMSS and the EUROHIS-QOL8 was only significant in patients who developed MF at V2 (N = 91; r = −0.277; p = 0.009) but not in patients who did not develop MF at V2 (N = 239; r = −0.111; p = 0.088). To develop MF after a 2-year follow-up was associated with an increase in the NMSS total score without controlling for other factors (β = 0.148; 95% CI, 2.69-16.98; p = 0.007) but also after adjustment to age, gender, years from symptoms onset, LEDD and the NMSS total score at baseline, and the change in LEDD from V0 to V2 as well (β = 0.128; 95% CI, 0.17-16.86; p = 0.046). However, when time on levodopa and the H&Y stage were included in the model as covariates, it was not significant (with time on levodopa therapy, p = 0.062; with H&Y, p = 0.167; both variables, p = 0.212). Development of MF was associated with an increase in the PDQ39SI from V0 to V2 (β = 0.135; 95% CI, 0.61-5.55; p = 0.015) but not with the change in the EUROHIS-QOL8 total score (p = 0.207). However, it was not significant after controlling for other covariates (age, gender, years from symptoms onset, LEDD and the PDQ-39SI at baseline, and the change in LEDD from V0 to V2) (p = 0.094). size = 0.16; p = 0.071) ( Table 2). The mean score on all domains of the PDQ-39SI was the highest in patients who developed MF after the 2-year follow-up at V2 and the lowest in patients who did not develop MF, at V0 (Figure 3). A moderate correlation was observed between the change from V0 to V2 in the NMSS total score and the change in the PDQ-39SI in the whole cohort (N = 320; r = 0.402; p < 0.0001) and in both groups, patients with (N = 91; r = 0.328; p = 0.002) and without MF (N = 239; r = 0.433; <0.0001) at V2. However, the correlation between the change in the total score of the NMSS and the EUROHIS-QOL8 was only significant in patients who developed MF at V2 (N = 91; r = −0.277; p = 0.009) but not in patients who did not develop MF at V2 (N = 239; r = −0.111; p = 0.088).

Discussion
The present study observes that MF are frequent in PD, appearing in a cohort of 330 patients with a mean of 4 years from symptoms onset in one of every 4 subjects after a 2-year follow-up, and also that they are related to NMS. Specifically, NMS burden was greater at baseline in PD patients who 2 years later developed MF, and the increase in the NMS burden after the 2-year follow-up was double in this group as well. Moreover, similar results were obtained in terms of QoL. Importantly, all patients at baseline were without MF and this is the first time that NMS burden progression is specifically analyzed regarding the development of MF in a PD cohort.
MF are frequent in PD [28][29][30][31]. In the COPPADIS cohort, of 690 patients with a mean disease duration of 5.5 years (DS 4.37), 33.9% had MF [17]. This percentage was 18.1% in the subgroup of patients with ≤5 years of disease duration (N = 396), with a mean disease duration of 2.7 years (DS 1.5) from symptoms onset [17]. The frequency will depend in part on the methods used-from an interview to wearable tools-and how sensitive we can be to detect them [32]. Stocchi et al. analyzed wearing-off (WO) in 617 PD patients with a mean disease duration of 6.6 years (DS 4.6) and observed that neurologist identified the presence of WO with an interview in 56.9% of the patients, whereas the percentage was 67.3% when the self-rated 19-question Wearing-Off Questionnaire (WOQ-19) was administered [33]. Identifying fluctuations is important in PD patients for two reasons. Firstly, their presence is associated with a worse status in terms of motor, NMS, QoL, and autonomy for ADL [17]. Secondly, the therapeutic strategy is conditioned by their presence to the point that there are several drugs marketed with indication to be only for patients with MF [34]. MF (either early or advanced) can significantly add to the NMS burden in PD [35,36]. However, few studies specifically focused on the NMS prevalence in motor-fluctuating PD patients [17,37,38]. Recent data published of 1589 PD patients from the SYNAPSIS study support the high prevalence of NMS in PD patients with MF in real-life condition, thus reinforcing the need for assessing them for diagnostic accuracy and for delivering holistic care [37]. Using the NMSS, we previously observed a greater NMS burden in the group with MF in a cross-sectional study conducted in PD patients from the COPPADIS cohort. In particular, 28 out of the 30 NMS included in the NMSS were significantly more frequent in patients with MF compared with those who did not have MF, and the mean score of all domains of the NMSS except urinary symptoms and sexual dysfunction was significantly higher in the group with MF [17]. Watanabe et al. recently explored the changes in NMS and QoL during 52 weeks in 996 Japanese PD patients exhibiting MF using the Movement Disorder Society Unified PD Rating Scale (MDS-UPDRS) Part I and the 8-item PD Questionnaire (PDQ-8), respectively [38]. They detected that changes in MDS-UPDRS Part I scores were variable and related to changes in HRQoL and identified 3 separate groups: unchanged (63.8%); deteriorated (20.1%); improved (16.2%). However, very importantly, all patients included in this study had MF. To our knowledge, our study is the first one to prospectively analyze the change in the NMS burden in relation to the development of MF in PD patients who initially did not have MF. As we previously reported in this cohort [39], about 6 out of 10 patients increased the NMSS total score after a 2-year follow-up. Although there were no differences in the percentage between the two groups-patients who developed MF and patients who did not develop MF-a greater NMS burden increase was observed in the first group. We did not analyze specifically if NMS fluctuated (e.g., NMS-MDS [40]), but this finding would support the relationship between NMS and the presence of OFF episodes with an increase in NMS perception during the OFF episodes. Importantly, the effect of MF on NMS burden persisted after adjustment to some variables related to NMS in PD such as age, gender, disease duration, or even dopaminergic medication [35,41,42]. However, NMS in PD are related to motor stage as well [17,18,25,42], and after the inclusion of the H&Y stage in the model, the effects disappeared. The same happened when time on levodopa therapy was included as covariate in the model. It is well-known that both aspects are related to the development of MF [30,31]. A more advanced H&Y stage is related to a greater degree of denervation of the striatal nucleus with more sensitivity to the development of MF [43]. On the other hand, a longer time on levodopa could imply a longer disease duration but also more time exposed to certain causative mechanisms (presynaptic and postsynaptic changes and pharmacokinetic and pharmacodynamic factors) [30,31]. The data as a whole indicate that PD patients who will develop MF in the short-term are patients with a more advanced disease with a greater NMS burden and patients with an increased risk of developing more severe NMS burden. To detect NMS burden progression is relevant because it is associated with a worse QoL [18,28]; importantly, in this context, MF development was associated with a greater worsening of both HRQoL and GQoL in the present analysis. To reduce NMS burden in PD patients has been demonstrated to be associated with an improvement in QoL [14,15]. In summary, our findings reinforce the idea that there is a close relationship between motor and NMS and that dopaminergic treatment can be helpful in some cases [5,10].
The present study has some limitations. The sample size of the group of patients with MF at V2 was smaller (N = 91) compared with the group without MF (N = 231), and the information about NMS burden follow-up was recorded in 330 patients of 462 (71.4%) without MF at baseline from the COPPADIS cohort. This is a limitation observed in other prospective studies, with percentages ranging from 61.9% to 89.8% [39,42,44]. We used the NMSS to assess the NMS burden progression, but some studies suggest that a battery of separate NMS scales is more sensitive to change than the NMSS [45]. Our sample was not fully representative of the PD population due to inclusion and exclusion criteria (i.e., age limit, no dementia, no severe comorbidities, no second line therapies, etc.). For some variables, the information was not collected in all cases (the smallest sample size was for the change in NPI (N = 255) since it was covered by the caregiver and not all had a primary caregiver). On the contrary, the strengths of our study include a very thorough assessment, a prospective longitudinal follow-up design, and the extensive clinical and demographic information recorded.
In conclusion, we demonstrated for the first time in a prospective study that, in PD, the development of MF is associated with a greater NMS burden increase in the short-term. In practice, it is essential to detect MF early and ask about NMS, especially in patients with a greater disease severity and a longer time on levodopa. has received honoraria for educational presentations and advice service by Abbvie, UCB Pharma, Zambon, Bial, and Teva. Vela L. has received honoraria for educational presentations and advice service by Abbvie, UCB Pharma, Lundbeck, KRKA, Zambon, Bial, and Teva. Escalante S. has received honoraria for educational presentations and advice service by Abbvie, Zambon, and Bial. Cubo E.; travel grants from Abbvie, Allergan, and Boston; and lecturing honoraria from Abbvie, International Parkinson's disease Movement Disorder Society. Carrillo Padilla F. has received honoraria from Zambon (SEN Congress assistance). Martínez Castrillo JC. has received research support from Lundbeck, Italfarmaco, Allergan, Zambon, Merz, and Abbvie; he has also received speaking honoraria from AbbVie, Bial, Italfarmaco, Lundbeck, Krka, TEVA, UCB, Zambon, Allergan, Ipsen, and Merz. Sánchez Alonso P. has received honoraria for educational presentations and advice service by Abbvie, UCB Pharma, Lundbeck, KRKA, Zambon, Bial, and Teva. Alonso Losada M. G. has received honoraria for educational presentations and advice service by Zambon and Bial. López Ariztegui N. has received honoraria for educational presentations and advice service by Abbvie, Italfarmaco, Zambon, and Bial. Gastón I. has received research support from Abbvie and Zambon and has served as a consultant for Abbvie, Exelts, and Zambon.

Informed Consent Statement:
Written informed consent from all participants in this study were obtained before the start of the study.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request. No computer coding was used in the completion of the current manuscript.