Nonmotor and diagnostic findings in subjects with de novo Parkinson disease of the DeNoPa cohort

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1 Nonmotor and diagnostic findings in subjects with de novo Parkinson disease of the DeNoPa cohort Brit Mollenhauer, MD* Ellen Trautmann, PhD* Friederike Sixel-Döring, MD Tamara Wicke, MS Jens Ebentheuer, MD Martina Schaumburg, MS Elisabeth Lang, BS Niels K. Focke, MD Kishore R. Kumar, PhD Katja Lohmann, PhD Christine Klein, MD Michael G. Schlossmacher, MD Ralf Kohnen, PhD Tim Friede, PhD Claudia Trenkwalder, MD On behalf of the DeNoPa Study Group Correspondence to Dr. Mollenhauer: ABSTRACT Objective: To determine nonmotor signs (NMS) and evaluate the utility of several diagnostic tools in patients with de novo Parkinson disease (PD). Methods: This is a large single-center study of the DeNoPa cohort, including frequency-matched healthy controls. This study covers motor signs, NMS, and a combination of diagnostic tests including olfactory testing, transcranial sonography of substantia nigra (TCS), and polysomnography (PSG). We report the frequency and characteristics of NMS and the outcomes of nonmotor tests at the time of diagnosis. Results: Cross-sectional analyses of baseline investigations identified significant differences in the NMS Questionnaire (NMSQuest) and the Scopa-AUT Gastrointestinal score in 159 drug-naïve PD patients vs 110 controls. In addition, patients with PD showed reduced olfactory function, hyperechogenicity on TCS, and higher frequency of REM sleep behavior disorder (RBD). In exploring predictive markers, we found that the combination of several investigations, i.e., the NMSQuest, Scopa-AUT Gastrointestinal score, and Smell Identification Test reached an area under the receiver operating characteristic curve (AUC) of (95% confidence interval [CI] ). With the addition of serum cholesterol and mean heart rate values, the AUC value reached (95% CI ); when TCS and PSG were added, the AUC increased to (95% CI ). Conclusions: We show feasibility and utility of standardized data acquisition in a large, single-center cohort of patients with de novo PD and matched healthy controls. The baseline results from our prospective investigations reached a value of.0.9 sensitivity and specificity for biological markers when we added routine laboratory investigations and quantified nonmotor features including sleep. Neurology â 2013;81: GLOSSARY AUC 5 area under the curve; CI 5 confidence interval; DeNoPa 5 De Novo Parkinson; NMS 5 nonmotor signs; NPH 5 normalpressure hydrocephalus; PD 5 Parkinson disease; PSG 5 polysomnogram; RBD 5 REM sleep behavior disorder; RBD-SQ 5 REM Sleep Behavior Disorder Screening Questionnaire; RBE 5 REM behavioral events; Scopa-AUT 5 Scale for Outcomes in PD for Autonomic Symptoms; TCS 5 transcranial sonography; UKBBC 5 UK Brain Bank Criteria; UPDRS 5 Unified Parkinson s Disease Rating Scale. Supplemental data at As of 2013, Parkinson disease (PD) is defined by the clinically recognized presence of motor signs and their responsiveness to levodopa (according to UK Brain Bank Criteria [UKBBC]). 1 Nonmotor signs (NMS), such as REM sleep behavior disorder (RBD), 2 constipation, 3 and olfactory dysfunction, 4 are evident during the course of PD 5,6 but are also known to be present in early disease stages. 7,8 Thus, it seems questionable whether the current clinical diagnosis of PD, based exclusively on motor features, is still appropriate. 1 At the time a patient is diagnosed, a considerable proportion of dopaminergic neurons of the substantia nigra have already degenerated, but other areas of the nervous system are also affected by intraneuronal Lewy body *These authors contributed equally to this work. From the Center of Parkinsonism and Movement Disorders (B.M., E.T., F.S.-D., T.W., J.E., M.S., E.L., C.T.), Paracelsus-Elena-Klinik, Kassel; Department of Neurosurgery (B.M., N.K.F., C.T.), University Medical Centre, Goettingen; Department of Psychology (E.T.), University Kassel; Section of Clinical and Molecular Neurogenetics at the Department of Neurology (K.R.K., K.L., C.K.), University of Luebeck; Departments of Neuropathology (B.M.) and Medical Statistics (T.F.), University Medical Centre, Goettingen, Germany; Program in Neuroscience (M.G.S.), Ottawa Hospital Research Institute; Division of Neurology, The Ottawa Hospital, University of Ottawa, Canada; and ReSearch Pharmaceutical Services (R.K.), Nuremberg, Germany. DeNoPa coinvestigators are listed on the Neurology Web site at Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article American Academy of Neurology

2 Figure 1 function that contain aggregates of misfolded a-synuclein. 9 These inclusions may spread trans-synaptically and thereby lead to manifestation of NMS prior to the classical PD motor symptoms 10 but data on established PD cases are sparse. 7,8,11,12 Thus, there is a need to systematically define NMS and diagnostic tools in established drug-naive PD subjects. The De Novo Parkinson (DeNoPa) study is a prospective longitudinal investigation of a single-center cohort comprising de novo PD subjects in early stages of the disease and healthy controls. The study encompasses clinical, genetic, neuropsychological, and ancillary investigations of NMS at baseline and biannual follow-up. The working hypotheses underlining our cross-sectional baseline assessment are 1) NMS are present and can be documented by existing questionnaires and established rating scales in a large cohort of de novo PD cases; and 2) objective diagnostic tools reveal significant differences compared to healthy controls. METHODS Objectives. We report the baseline data from the longitudinal DeNoPa cohort including the comparison with a group of frequency-matched healthy controls in cross-sectional analyses. Recruitment was completed over 29 months between September 2008 and January 2012 (recruitment for controls started in February 2009) at a special clinic for early PD patients located at the Paracelsus- Elena Klinik (Centre of Parkinsonism and Movement Disorders) in Study tree of screened and enrolled patients and healthy controls Kassel, Germany. The clinic is the nation s largest hospital in Germany solely dedicated to the care of patients with parkinsonism and is being utilized by a network of neurologists within the city s catchment area and beyond (appendix e-1 on the Neurology Web site at Recruitment. During the recruitment period, we specifically asked the referring neurologists to send de novo PD subjects for a thorough clinical evaluation in our inpatient hospital. Healthy controls were recruited through relatives and friends of the enrolled subjects and other patients of our clinic as well as through a newspaper advertisement in early We offered a free health check for all controls (for details, see appendix e-2). Study participants. Screening was first performed by a neurologist specializing in movement disorders (B.M., C.T., or J.E.). Subjects meeting the inclusion criteria were evaluated by an independent second movement disorder specialist (B.M., C.T., or F.S.-D.) and received a standardized program of investigations (figure 1, table e-1). Subjects had to be between 40 and 85 years old, newly diagnosed with PD featuring at least 2 of resting tremor, bradykinesia, and rigidity according to UKBBC, 1 and had to fulfill de novo criteria with L-dopa exposure no longer than 2 weeks and not within 4 weeks prior to study entry. Exclusion criteria were 1) known severe vascular encephalopathy or normal-pressure hydrocephalus (NPH) as shown on MRI when available at screening or when detected during imaging studies or 2) signs or symptoms according to multiple system atrophy or progressive supranuclear palsy (according to consensus criteria 13,14 ), or medication-induced PD. Healthy controls had to be between 40 and 85 years old, without any active known/treated condition of the CNS and without a family history of idiopathic PD. Antipsychotic drugs were an exclusion criterion both for controls and patients. Controls were matched using frequency matching 15 by age, sex, and education. The diagnosis was established according to UKBBC with the investigators blinded to the outcome of NMS assessments. Investigations. Demographic variables and medical history, including current comorbidities and medication, were assessed by interview (table 1). A neurologist and internist examined all patients. To exclude a genetic cluster that may have arisen due to regional recruitment, we performed a genetic analysis of the most frequently observed PD gene mutations in a Central European population (table e-2). We assessed NMS using validated questionnaires covering gastrointestinal symptoms, pain, sleep, mood, and quality of life. A selection of neuropsychological tests was based on their use and recommendation in patients with PD 16,17 (all references of questionnaires and tests are given in table e-1). For further investigations, we assessed olfactory function using Sniffin Sticks, performed routine ECG in a quiet room, obtained selected laboratory and blood parameters, carried out a 2-night video-polysomnogram (PSG), and obtained imaging by transcranial sonography (TCS) of the substantia nigra and 1.5-T MRI of the brain (for details, see appendix e-2). *Reasons for exclusion in the patient group were as follows: decline of study participation (n 5 36), prior Parkinson disease medications received (n 5 39), signs or symptoms for other (atypical) disease (n 5 111). In healthy controls: positive family history for neurodegenerative disease (n 5 28) or current/previous neurologic condition (n 5 2). **A total of 52 interested controls were not enrolled in accordance with the matching criteria (especially age and education). MMSE 5 Mini-Mental State Examination; NPH 5 normal-pressure hydrocephalus. Statistical analysis. The x 2 test, Student t tests, and analyses of covariance or logistic regression (for dichotomous variables) were used to examine differences in baseline data after multiple imputation (for missing values). Furthermore, we performed receiver operating characteristic analyses to determine optimal cutoff points of diagnostic tools (including cross-validation for bias correction in area under the curve [AUC] estimation). Detailed information on the statistical analysis can be found in appendix e-2. Neurology 81 October 1,

3 Table 1 Demographic data of subjects with PD and healthy control subjects and clinical data and motor assessments of patients with PD PD (n 5 159) HC (n 5 110) p Value Age, y (58.00/73.00) 65.5 (60.00/70.00) 65.3 (9.70, ) 64.7 (6.83, ) a Male/female (%) 105/54 (66/34) 67/43 (60/40) a Number of comorbidities 2.0 (1.00/4.00) 3.0 (0/3.00) 2.6 (1.70, 0 8) 2.7 (1.62, 0 7) Number of medications b 2.0 (1.00/4.00) 1.0 (0/3.00) Symptom duration, mo 15.0 (9.00/24.00) 3.0 (2.61, 0 12) 1.8 (1.91, 0 9), (37.11, ) UPDRS total score c 28.0 (17.00/39.00) 0 (0/1.00) UPDRS motor score before levodopa n (14.67, ) 0.9 (1.49, 0 8), (11.00/26.00) 19.0 (10.31, ) Abbreviations: HC 5 healthy control subjects; PD 5 Parkinson disease; UPDRS 5 Unified Parkinson s Disease Rating Scale. Data are median (25th/75th centiles), mean (SD, range), or number (%). a Age and sex were part of the frequency matching. b Number of neuroleptic medications ever taken was 5 in subjects with PD and none in HC. c Movement Disorder Society UPDRS revealed scores of (7 88) for subjects with PD. Standard protocol approvals, registrations, and patient consents. We conducted the study according to the Declaration of Helsinki and all subjects provided informed written consent. The ethics committee of the Physician s Board Hesse, Germany (approval no. FF89/2008) approved the study, which is registered at the German Register for Clinical Trials (DRKS ) according to the WHO Trial Registration Data Set. RESULTS We assessed the eligibility of 491 subjects, and subsequently included 162 patients and 114 controls in the study (figure 1). A scheduled follow-up review of enrolled participants revealed that a total of 7 subjects had to be excluded from the study due to the following reasons: occurrence of 1) predominant postural tremor and polyneuropathy (n 5 1, patient group), 2) features of NPH in MRI not present at screening (n 5 2, patient group), and 3) presence of cognitive decline on Mini-Mental State Examination scores (,26) (n 5 4, control group) (figure 1). Demographic data are shown in table 1. Genetic analysis. Genetic analysis revealed 6 PD patients with heterozygous Parkin mutations (3.8%) but no known mutation was detected in PINK1, DJ-1, SNCA, or LRRK2. Heterozygous GBA1 variants were found in 3 patients but in none of the controls. Frequencies of the investigated risk variants in SNCA and MAPT are shown in table e-2. Motor symptoms. Motor symptoms of the PD group by the Unified Parkinson s Disease Rating Scale (UPDRS) are shown in table 1. Nonmotor features and ancillary investigations are described below (and given in tables 2 and 3). Nonmotor features. NMS were reported more often in patients with PD than in controls by self-rating questionnaires using the global PD NMS tools (NMSQuest and -scale; p, 0.001) 18,19 as well as the Scopa-AUT score 20 for gastrointestinal and cardiovascular dysfunction (p, 0.001). Detailed assessment of sleep-related changes showed differences between patients and controls as assessed by 2 questionnaires: Parkinson s Disease Sleep Scale version 2 21 and REM Sleep Behavior Disorder Screening Questionnaire (RBD-SQ 22 ;bothp, 0.001). The Parkinson s Disease Questionnaire 39 total score 23 was elevated in subjects with PD compared to controls (p, 0.001) and correlated significantly with both the motor (UPDRS III) and nonmotor symptoms (by NMSQuest; p, 0.001, respectively) (table 2). Ancillary investigations. Subjects with PD performed worse in all 3 subtests of the olfactory tests (smell threshold, odor discrimination, and stimulus identification, p, 0.001) (table 3). We selected the identification test for further analysis because it is easier to conduct, faster to perform, and showed the best discrimination (data not shown). Routine ECG revealed higher values for mean heart rate and shorter QT interval (not shown) in patients with PD (p, 0.001; table 3). Any treatment affecting heart rate (b-blockers, calcium channel blockers, and glycosides) or blood pressure did not 1228 Neurology 81 October 1, 2013

4 Table 2 Subjective nonmotor features of de novo PD patients and healthy controls assessed by validated self-rating scales PD (n 5 159) HC (n 5 110) Mean difference (95% CI) p Value Unadjusted p value NMSQuest n n 5 110,0.001, (4.05, ) 3.7 (2.54, ) (24.63, 22.97) NMSS n n 5 110,0.001,0.001 Scopa-AUT n n (30.72, ) 13.1 (13.38, ) (230.30, ) Gastrointestinal 2.7 (2.30, ) 0.7 (1.08, ) (22.41, 21.50),0.001,0.001 Urinary 5.0 (3.37, ) 4.1 (2.84, ) (21.66, 20.17) Cardiovascular 0.7 (0.92, 04.00) 0.17 (0.45, ) (20.69, 20.31),0.001,0.001 Thermoregulatory 1.8 (1.84, ) 1.3 (1.62, ) (20.95, 20.10) Pupillomotor 0.6 (0.79, ) 0.3 (0.72, ) (20.44, 20.07) Sexual dysfunction, man 1.9 (2.01, ) 1.2 (1.65, ) (21.35, 20.20) Sexual dysfunction, woman 0.9 (1.31, ) 0.78 (1.19, ) (20.68, 0.48) PDQ-39 total score n n 5 73,0.001, (10.36, ) 3.6 (3.76, ) (212.31, 27.85) PDSS total score n n 5 107,0.001, (8.55, ) 10.2 (6.34, ) (27.23, 23.40) RBD-SQ n n 5 92, (2.8, ) 2.2 (2.1, ) (22.305, ) RBD-SQ cutoff (>5), yes/no (%) n n /87 (30/70) 12/79 (15/85) 2.67 (2.01, 3.56) Abbreviations: CI 5 confidence interval; HC 5 healthy control subjects; NMSQuest 5 Nonmotor Symptoms Questionnaire; NMSS 5 Nonmotor Symptoms Scale; PD 5 Parkinson disease; PDQ-39 5 Parkinson s Disease Questionnaire 39; PDSS-2 5 Parkinson s Disease Sleep Scale version 2; RBD-SQ 5 REM Sleep Behavior Disorder Screening Questionnaire; Scopa-AUT 5 Scale for Outcomes in PD for Autonomic Symptoms. Data are mean (SD, range), mean difference (95% CI), and p values with multiple imputation and adjustment for age, sex, and education. show an effect on the significance of changes in heart rate by regression analysis (p. 0.05). Routine blood values showed higher values for mean corpuscular hemoglobin (p ) and lower values for total fasting cholesterol (p, 0.001) in PD but the latter only applied to male patients older than 55 years (table 3). There were no group differences for other blood values. In particular, the endogenous neuroprotectant and antioxidant, plasma urate, was not significantly different between those with PD and controls (p ). TCS revealed larger hyperechogenic areas of the substantia nigra in subjects with PD (p, 0.001; table 3), as expected from previously published reports. PSG identified 17 (15%) healthy controls and 81 (51%) subjects with PD with evidence of abnormal motor behaviors in REM, defined as REM behavioral events (RBE) (p, 0.001; table 3). For the definition of RBE, see appendix e-2. RBD severity measured with the RBD Severity Scale showed no differences between the groups (p ). Only REM sleep latency was longer in the PD group (p ); sleep stages, REM duration, and all other PSG parameters showed no difference between patients and controls. The RBD-SQ identified only 30% of RBE subjects among the PD cases (and 15% in controls according to cutoff values of 5 points), 22 and was therefore deemed to be insufficient compared to formal PSG evaluation (table 3). Ancillary test steps. When probing for the specificity and sensitivity of individual and aggregate scores (table e-3) relatedtonmssymptomsandsigns,weobtainedthe following results: the analyses of the NMS-Quest and the Scopa-AUT Gastrointestinal subtest revealed an AUC of (confidence interval [CI] ; step 0). When the Smell Identification Test was added, the AUC reached (CI ; step 1; figure 2A). Furthermore, with serum cholesterol and heart rate (step 2; figure 2A) added, it increased the AUC slightly to (CI ). When results from additional investigations complemented the test battery, the AUC reached (CI ) with hyperechogenicity of substantia nigra from TCS (step 3; figure 2A); with RBE identified by PSG (step 4; figure 2A) entered as additional variable, it climbed to (CI ). The introduction of a cognitive screening instrument (e.g., the clock-drawing test) at any level did not improve the diagnostic performance. Neurology 81 October 1,

5 Table 3 Technical investigations: Olfactory test, ECG, serum cholesterol, presence of REM sleep behavior disorder in polysomnography, and transcranial sonography PD (n 5 159) HC (n 5 110) Olfactory testing n n Mean difference (95% CI) p Value Unadjusted p value Threshold 3.1 (3.62, ) 7.1 (3.69, ) 3.89 (3.01, 4.77),0.001,0.001 Discrimination 8.3 (3.52, ) 12.0 (2.52, ) 3.63 (2.87, 4.38),0.001,0.001 Identification 7.2 (3.52, ) 12.0 (2.65, ) 4.70 (3.93, 5.47),0.001,0.001 ECG n n Heart rate (1/min) 68.9 (11.34, ) 61.5 (9.36, ) (210.16, 25.17),0.001,0.001 Total serum cholesterol (mg/dl) n n PSG (39.97, ) (40.27, ) (10.11, 29.63),0.001,0.001 RBE yes/no (%) 81/77 (51/49) 17/93 (15/85) 5.72 (4.46, 7.33),0.001,0.001 RBD yes/no (%) 40/118 (25/75) 2/108 (2/98) ( ),0.001,0.001 Transcranial sonography a n n Substantia nigra Echogenicity Bilateral mean (cm 2 ) (0.20, ) (0.07, ) (20.18, 20.10),0.001,0.001 Abbreviations: CI 5 confidence interval; HC 5 healthy control subjects; PD 5 Parkinson disease; PSG 5 polysomnogram; RBD 5 REM sleep behavior disorder; RBE 5 REM behavioral events. Data are mean (SD, range), number (%), or mean difference (95% CI), and p values with multiple imputation and adjustment for age, sex, and education. a Only performed in sufficient bone window. To facilitate clinical application in the future, we explored a test battery that was divided into 3 arms depending on the availability of investigations: Option 1: Step 2 (questionnaires, olfaction, ECG, and serum cholesterol) for easy screening purposes (AUC 0.913; CI ). Option 2: Step 2 (questionnaires, olfaction, ECG, and serum cholesterol) plus TCS for wide application and when ultrasound is available (AUC 0.957; CI ; figure 2B). Option 3: Step 2 (questionnaires, olfaction, ECG, and serum cholesterol) plus PSG for smaller cohorts when PSG is available (AUC 0.921; CI ; figure 2C). DISCUSSION To investigate nonmotor features and biomarkers for PD, we studied 159 early de novo PD patients and 110 healthy control subjects using assessments of NMS and tools previously adapted to identify patients with PD. We found that changes in nonmotor symptoms assessed by the NMSQuest and subscores of the Scopa-AUT scale as well as measures of olfaction, heart rate, and sleep were the most common nonmotor findings in early PD. The suggested combinations provided a tool to diagnose early disease state with an AUC value of up to for our group of de novo PD patients who had a mean UPDRS motor score of 19.0 (reflecting an early stage of disease, as measured by their motor deficits). Studies on NMS in early de novo PD subjects are currently sparse: 1. The NMSQuest was significantly increased in 46 de novo PD subjects and healthy controls independently of dopaminergic medication. 24 The NMSQuest could possibly be used as a screening tool for further selecting patients for more expensive investigations (like PSG). 2. Olfactory dysfunction affects up to 90% of patients with PD and has been shown to be a marker candidate for the premotor stage of the disease. 25 In a smaller de novo PD cohort, olfactory dysfunction was shown in 24 (out of 41) untreated patients with PD and 18 (out of 24) healthy controls. 26 A possible association between olfactory dysfunction and the development of early PD has been suggested by several groups. 27,28 3. Abnormal parasympathetic, but not sympathetic, cardiac dysfunction at an early disease stage has been shown in 7 patients with PD and 7 healthy controls, 29 and impaired heart rate variability was suggested as an early diagnostic tool 30 and thought to be related to Lewy body pathology within cardiac neurons. 10 In our study, we found higher heart rate and lower cholesterol levels in our subjects with PD. Lower serum cholesterol was previously reported to occur in patients with PD, independent of nutritional status and body mass index. 31,32 Dysregulation of cholesterol trafficking was shown to be 1230 Neurology 81 October 1, 2013

6 Figure 2 Cumulative receiver operating characteristic curves with area under the curve of the selected tests Cumulative receiver operating characteristic (ROC) curves with area under the ROC curve (AUC) of the selected tests in (A) different steps and (B, C) 2 different options depending on availability at sites. Chosen combinations: step 0: questionnaires only: Nonmotor Signs Questionnaire 1 Scopa-AUT Gastrointestinal; step 1: step 0 plus Smell Identification Test; step 2: step 1 plus serum cholesterol 1 ECG (heart rate); step 3: step 2 plus transcranial sonography (TCS) (substantia nigra); step 4: step 3 plus REM sleep behavioral events. CI 5 confidence interval; PSG 5 polysomnogram. Neurology 81 October 1,

7 involved in the pathogenesis of neurodegeneration in PD and an interaction with SNCA was proposed. 33 Our findings on elevated mean heart rate evaluated by routine ECG are new. They need to be interpreted with caution and validated in independent, large-scale studies. 4. Hyperechogenicity of substantia nigra by TCS is present in several early PD studies and in one follow-up investigation 34 and in 52% of our patients. Comparedtohealthycontrols,werecordedonlyarate of 10% for hyperechogenicity. Whether these subjects develop a neurodegenerative disease in the future will be investigated in follow-up studies. Because TCS was carried out in an open (unblinded) fashion, tends to be observer-dependent, and has not been internationally employed as a tool, we offered option 3, comprising step 2 plus RBE without TCS (reaching an AUC of 0.921). 5. Presence (or absence) of RBE, a new method as assessed by video PSG, 35 has not been investigated so far in a large cohort of de novo PD patients and controls. Here, we identified a significantly higher prevalence of movement events in REM sleep (51%) in patients with early PD compared to controls (15%), not yet fulfilling the entire spectrum of violent RBD or classification of RBD according to International Classification of Sleep Disorders. 36 Numerous reports on the association of RBD with definite PD have been published, suggesting that RBD reflects an underlying a-synuclein related disorder starting in the prodromal phase of PD. In fact, up to 82% of older men diagnosed with RBD develop parkinsonism/dementia. 37,38 The findings we report here have 2 major implications. First, they suggest that the current clinical criteria of PD 1 miss the full spectrum of clinical symptoms and signs in the early stages of PD with evolving motor symptoms, which likely reflect the nervous system wide process of typical PD. 39,40 Second, it is evident from our study that the crosssectional assessment of subjects with NMS requires an interdisciplinary approach in order to make an accurate diagnosis of PD. We are aware of several limitations of our study, which include the following: 1. Healthy controls were recruited through newspaper advertisements and represent a population that is well aware of health concerns and thus more inclined to support research. 2. Most tests were carried out in an open, semiblinded, or unblinded fashion, such as TCS, parts of PSG, and olfactory testing. Blood analysis and examination of heart rate were investigator-blinded. 3. Despite the fact that we have excluded 111 subjects from our screening cohort of 348 patients with additional neurologic signs, i.e., in accordance with criteria to suggest multiple system atrophy or progressive supranuclear palsy, we may have included several patients with atypical Parkinson syndrome at early stage of their illness. 4. Our results originate from a single cohort, and we need to now validate our proposed screening combination and delineated AUC values in an independent, early population of patients with PD. Whether these marker candidates can also measure disease progression or act as predictors of distinct subtypes (endophenotypes) down the road (i.e., PD) among subjects with a shared motor phenotype at the time of enrolment will be investigated during already initiated follow-up investigations in our DeNoPa cohort. AUTHOR CONTRIBUTIONS B.M., F.S.D., and C.T. designed the study and were responsible for recruitment and data processing. E.T. and T.F. oversaw all statistical analyses. T.W., J.E., E.T., M.S., E.L., and N.K.F. were responsible for performing investigations and collecting and entering data. K.R.K., K.L., and C.K. performed genetic analyses and assisted in data interpretation. N.K. and M.G.S. were involved in sample analyses and data interpretation. C.T. oversaw patient recruitment and characterization and assisted in the interpretation of data. B.M., C.T., and E.T. wrote the manuscript. F.S.-D., M.G.S., T.F., C.K., R.K., and K.L. coedited the manuscript. B.M., C.T., and F.S.-D. had full access to the clinical primary data. B.M. and E.T. have full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors had access to the data generated in the study including the statistical analysis and decided to submit the paper for publication. ACKNOWLEDGMENT The authors thank all participants in the study and their families as well as referring neurologists; Bernard Ravina for excellent advice; Maria Rudolph and Miriam Schanze for support with data entry; and Anne-Marie Williams for help in editing the language of the manuscript. STUDY FUNDING Supported by unrestricted research grants from the Paracelsus-Elena-Klinik, Kassel, Germany, and unrestricted research grants from TEVA Pharma/ Lundbeck, GE Health care, and the Hermann und Lilly Schilling Foundation. The study sponsors provided support through an unrestricted grant and had no influence on the study design, collection and analysis of data, the writing of the paper, or the decision to submit the paper. The sponsors have been informed about the final manuscript and the submission for publication. DISCLOSURE Dr. Mollenhauer has received speaker honoraria from Orion Corporation and GlaxoSmithKline; serves as an Associate Editor for the Journal of Alzheimer Disease; holds or has pending patents re: Method of differentially diagnosing dementias, Novel ELISA-based quantification of a-synuclein proteins in CSF and peripheral blood products using 384-well plates and MicroRNA expression profiling of CSF. She serves as a consultant for Bayer Schering Pharma AG and receives research support from Teva Pharmaceutical Industries Ltd., Desitin Pharmaceuticals, GmbH, Boehringer Ingelheim, GE Healthcare, the Michael J. Fox Foundation for Parkinson s Research, the American Parkinson s Disease Association, the Stifterverband für die Deutsche Wissenschaft (Dr. Werner Jackstädt-Stipend), and BMBF. Dr. Trautmann reports no disclosures. Dr. Sixel-Döring serves/has served on scientific advisory boards for Orion Corporation and Medtronic, Inc.; has received funding for travel from Boehringer Ingelheim; and has received 1232 Neurology 81 October 1, 2013

8 speaker honoraria from Boehringer Ingelheim, Cephalon, Inc., Medtronic, Inc., Solvay Pharmaceuticals, Inc., UCB, and Orion Corporation. T. Wicke, Dr. Ebentheuer, M. Schaumburg, and E. Lang report no disclosures. Dr. Focke received funding from the Stifterverband für die Deutsche Wissenschaft (Dr. Werner Jackstädt-Stipend); served in an advisory board for GE Healthcare; and received honoraria from GlaxoSmithKline and travel support from UCB/Schwarz Pharma. Dr. Kumar has received a Movement Disorders Society Travel Grant (2012) and is a recipient of the National Health and Medical Research Council (NHMRC) of Australia Dora Lush Postgraduate Scholarship. Dr. Lohmann receives funding from the German Research Foundation. Dr. Klein is a member of the editorial board of Neurology and has served as editor of the Continuum Issue Neurogenetics 2008 and as faculty at the annual meetings of the American Academy of Neurology since She has received consulting fees from Boehringer Ingelheim and Centogene and has received honoraria for speaking from Boehringer Ingelheim and Merz Pharma. She is the recipient of a career development award from the Hermann and Lilly Schilling Foundation. She is funded by the Volkswagen Foundation, the Deutsche Forschungsgemeinschaft, the Possehl Foundation, and received institutional support from the University of Luebeck for genetics research. Dr. Schlossmacher has received grants from the Michael J. Fox Foundation for Parkinson s Research, the Parkinson Research Consortium Ottawa, and the Government of Canada (CRC/CFI) and has served as an ad hoc, paid consultant for FoldRx, Genzyme Inc., Johnson & Johnson, Amicus Therapeutics, Elan Pharmaceuticals, Novartis Inc., and LINK Medicine. He has received lecture honoraria from Teva Neuroscience and has a scientific collaboration with Covance Inc, Epitomics Inc, and the Michael J. Fox Foundation. Dr. Kohnen has served on scientific advisory boards for UCB, Pfizer, and Mundipharma International Limited. T. Friede is an Associate Editor of Biometrical Journal and BMC Medical Research Methodology, a member of the Editorial Board of Therapeutic Innovation & Regulatory Science (formerly known as the Drug Information Journal ), and an Academic Editor of PLOS One. He has acted or acts as a consultant to Novartis Pharma AG, Pharmalog Institut für Klinische Forschung GmbH, Biogen Idec, CSL Behring, Tibotec, and Schering Plough (Merck). He has received research funding from Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, and the Executive Agency for Health and Consumers. Dr. Trenkwalder serves on scientific advisory boards for Teva Pharma, Boehringer Ingelheim, UCB, Novartis, Mundipharma International Limited, and Britannia. She has received speaker honoraria from Boehringer Ingelheim, UCB, Novartis, and GlaxoSmithKline and serves on the editorial board and as guest editor of Sleep Medicine, CNS Drugs, and Movement Disorders. She receives research support from Teva Pharmaceutical Industries Ltd. and Mundipharma International Limited. Go to Neurology.org for full disclosures. Received March 28, Accepted in final form July 1, REFERENCES 1. Hughes AJ, Daniel SE, Blankson S, Lees AJ. A clinicopathologic study of 100 cases of Parkinson s disease. Arch Neurol 1993;50: Postuma RB, Lang AE, Massicotte-Marquez J, Montplaisir J. 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