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

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Online-Only Supplementary Material (Mollenhauer et al.)

Appendix e-2

Assessment of motor function
Motor symptoms were assessed with the Unified Parkinson Disease Rating Scale (UPDRS)1 including Hoehn and Yahr (severity) scale2 and a standardized levodopa challenge test was performed in all PD patients.

Genetic analysis
Genetic analysis in PD patients comprised multiplex ligation-dependent probe analysis (MLPA; P051 probe mix, MRC Holland) to test for exon rearrangements in α-Synuclein (SNCA), Parkin, PINK1, and DJ-1 as well as for G2019S in LRRK2, and A30P in SNCA. In individuals found to carry an exon rearrangement in the Parkin gene in the heterozygous state, all 12 Parkin exons were sequenced. In addition, we screened for GBA mutations in exons 8 to 11 by sequence analysis and tested for known genetic risk factors of PD including Rep1 and rs11931074 in SNCA, and H1/H2 in MAPT in patients and controls.

Standardized levodopa testing
To test response to dopaminergic medication (as a criterion of the UK Brain Bank Criteria) and to identify symptoms which respond to dopaminergic therapy for future reference, we carried out standardized levodopa testing in PD patients. 250 mg levodopa was administered under fasting conditions (in the morning between 8 and 9 o’clock) after pre-treatment with domperidone 20 mg, 12 and one hour before assessment.3 UPDRS part III (motor score)1 and MDS-UPDRS4 were performed by one certified neurologist (BM or JE) before and one hour after administration, and documented by video recording.
All other investigations were carried out in PD patients before a levodopa challenge test was performed to avoid effects of dopaminergic medication.

Scales
Since we hypothesized the presence of NMS in early, de novo PD subjects5 we included established scales for known NMS: Assessment of autonomic dysfunction in Parkinson’s disease (Scopa-AUT)6, Parkinson’s Disease Questionnaire (for Quality of life ; PDQ-39)7, Non-motor Symptoms Questionnaire (NMSQuest) and –Scale (NMSS)8-10, Parkinson’s Disease Sleep Scale version 2 (PDSS-2)11, Epworth Sleepiness Scale (ESS)12, 13, Medical Outcomes Study Sleep Scale (MOS-Sleep)14, REM Sleep Behavior disorder Screening Questionnaire (RBD-SQ)15, REM-Sleep-Behavior Disorder Severity Scale (RBD-SS).16
All questionnaires have been applied to PD subjects and controls. In case of PD-specific questionnaires (PDQ-39) these were adapted for the healthy controls (by omitting “with respect to your PD…”).

Neuropsychological testing
Since dementia has been shown to be evident in advanced PD17 we hypothesized neuropsychological deficits, i.e. in the executive functions could be detected already early. All subjects were therefore tested by board-certified psychologists who were trained in neuropsychological testing (ET, MS). Cognitive screening instruments for assessing global efficiency were applied,18, 19 as well as tests for specific cognitive domains (executive functions, attention and speech,20-23 memory,24-26 visuospatial function27), psychiatric aspects and emotional stress by self-evaluation and partner interviews (Table e-1).28-32

(1) Olfactory testing
PD related α-synuclein pathology is present in the olfactory bulb early during the disease course and olfactory dysfunction is often reported to be present years before the motor symptoms appear.33 Olfaction was therefore tested using “sniffin’ sticks” (Burghart Medizintechnik GmbH, Wedel, Germany).34, 35 This test battery comprising 120 felt-tip pens (sticks) with 4 mL of odorant dissolved in propylene-glycol was used according to described instructions for three independent tests.35
The “perception threshold discrimination” test is a 1:2 dilution series with 16 stages starting with 4% dilution of the odorant. The “olfactory threshold” is determined using the single staircase method described by Doty.36 The “odor discrimination test” is a forced multiple-choice test comprising 16 odors, which should be recognized among a list of four possibilities given.37 All subjects performed all three tests.

(2) Electrocardiogram
Parasympathetic, but not sympathetic, cardiac dysfunction at early stages has been shown in only few PD subjects compared to healthy controls38 and impaired heart rate variability was suggested as an early diagnostic tool39 and related to cardiac Lewy body pathology.40 We therefore hypothesized early findings even in routine ECG. Routine 12–lead electrocardiogram (ECG) was carried out with a MAC 1200 ST by GE Healthcare and the following information extracted for analysis: Heart rate, QRS complex, QT interval and the PR segment.

(3) Biological fluid collection and analysis
Marker studies based on biological fluids on early de novo PD subjects have been sparse and mostly focused on single markers or indicators for developing a valid diagnostic procedure for PD. Lower serum cholesterol was previously shown in PD patients, independent from nutritional status and body mass index.41, 42 Dysregulation of cholesterol trafficking was shown to be involved in the pathogenesis of neurodegeneration in PD including an interaction with SNCA.43 Based on the current literature we further on hypothesized decreased hematological and uric acid levels early PD.44 Blood was consistently collected with BD Vacutainer® system tubes (BD, Franklin Lakes, NJ, USA) by venous puncture and processed according to published standard operating procedures (SOPs)45 in the morning between 7 and 9 o’clock after 12 hours fasting. Aliquots were stored at -80˚celsius within 30 minutes after the venous puncture.

To exclude other diseases we analyzed a panel of routine blood values: red- and white blood cell count, HGB, HCT, MCH, MCHC, PLT, aGot, aGPT, aGT, AlkPhos, Bilirubin, BUN, uric acid, total cholesterol, triglycerides, glucose, C-reactive protein, thyroid stimulating hormone (TSH basal), partial thromboplastin time, reticulocytes. Uric acid was investigated because of previously known changes in PD, TSH because of tremor being influenced by thyroid hormones.

We also collected cerebrospinal fluid (CSF) according to SOP45 but did not include the analysis of CSF in the current publication. The CSF data will be used primarily on prospective follow-up analysis for disease progression.

(4) Polysomnography (PSG)
Patients and healthy controls were studied in the sleep laboratory for two nights. Video-supported polysomnography (vPSG) was carried out as published elsewhere.46 The second night was used for analysis. If a study subject refused the second nighttime recording, or one of the two vPSGs was invalid due to technical artifacts, the valid recording was used. Motor behaviors and/or vocalizations with a purposeful component other than comfort moves were identified on the time-synchronized video as REM sleep behavioral events (RBE) by experienced raters (FSD & CT). Two or more events had to be present to be classified as “RBE positive”. RBE subjects included REM behavior disorder (RBD) and non-RBD subjects based upon the presence or absence of a specific amount of REM without atonia (RWA). Diagnosis of definite RBD was assumed according to criteria established by Schenck and colleagues47 and the International Classification of Sleep Disorders, 2nd edition (ICSD 2).48 Further details on classification of motor events in REM sleep and techniques for measuring RWA are reported elsewhere [new reference: Sixel-Döring F, Trautmann E, Mollenhauer B, Trenkwalder C: Rapid eye movement sleep behavioral events: A new marker for neurodegeneration in early Parkinson disease? SLEEP (in press 2013)].

(5) Transcranial sonography (TCS)
Transcranial sonography (TCS) showing hyperechogenicity of substantia nigra related to neurodegeneration has been shown to be present in advanced and early PD cases49 and indicates neurodegeneration in the premotor stages before the evidence of motor symptoms.50 We therefore performed TCS using a phased-array ultrasound system equipped with a 1.82 MHz transducer (Acuson Antares 5.0, Siemens, Erlangen, Germany). Through a preauricular acoustic bone window (penetration depth of 15 cm; dynamic range of 40 dB) width of the third ventricle was measured and the substantia nigra (SN) identified within the butterfly-shaped structure of the mesencephalic brainstem as clearly as possible. The area of echogenic signals was analyzed and quantified according to van de Loo51 and our own cut-off values (Table e-3)

(6) Magnetic resonance imaging (MRI)
To assess structural brain abnormalities and to enable further investigations (iron deposition, vascular changes and volumetric assessment) hypothesizing early atrophic changes in PD we conducted MRI on a Philipps Archiva 1.5 Tesla scanner in all participants (without contra indication such as pace maker). The scanning paradigm included (1) 6 mm transversal T2-FLAIR [repetition time (TR): 6000 ms, echo time (TE) 120 ms); (2) transversal T2 (6mm; TR 4800 ms; TE 110 ms); (3) fast field echo T2 (T2*) (6mm; TR 605 ms; TE 23 ms, flip angle 18˚) as well as (4) sagittal turbo far field T1 (1mm; TR 7.5 ms; TE 3.5 mm, flip angle 8°). In 25 subjects MRI was not performed due to contra indications (i.e., pace marker, severe claustrophobia) but computed tomography was carried out instead when possible.

Recruitment and retention strategies comprise regular subject meetings; regular newsletters were mailed every 3-4 months and web-based information is available (www.denopa.de; in German).

The more detailed results of the PSG investigation, as well as the analysis of cerebrospinal fluid, neuropsychological tests and MRI will be reported in separate manuscripts.

Detailed statistical analysis
Comparison of baseline characteristics of PD patients and HC were performed as t-tests (Table 1). Further significance testing (Tables 2 and 3, Table e-3) of between-group differences (PD, HC) was done by analysis of covariance (ANCOVA) with the covariables age, sex and education used in the group matching strategy. For dichotomous variables logistic regression analysis was carried out. Assumptions made in regression modeling were assessed by standard diagnostics (e.g. residual plots).52 We report mean difference, 95% confidence interval (CI) and p-values of the adjusted analyses in the tables and p-values from unadjusted analyses of variance and logistic regression (in the Tables 2, 3). We carried out receiver-operating characteristic (ROC) analyses to determine optimal cut-off points for continuous variables and report the area-under-the-ROC curve (AUC) including the 95% CI (Table e-3). To minimize positive bias in AUC estimation due to over fitting caused by estimating the AUC on the same data as developing the model the AUC estimates were bias corrected using cross validation.53

In addition we calculated sensitivity preferred strategies, which are more appropriate for screening tests (Table e-3).54 Due to the case-control character of our study we did not calculate predictive values. Screening test batteries were developed by combining diagnostic tests with high AUC into risk scores, which were derived from logistic regression models. Effects associated with p-values smaller than 5% are referred to as statistically significant. Due to the exploratory nature of this study the analyses were not adjusted for multiplicity. Subsequent analyses focused on a stepwise algorithm of selected investigations for diagnosing PD.
Missing data were partially caused by the insufficiency of some questionnaires (e.g. the RBD questionnaire includes questions about the behavior during the night and no possibility for e.g. not ascertained/not known is given) but could also be due to the lack of motivation caused by the large numbers of questions, although spread within several days. We therefore carried out a multiple imputation generating five complete data sets.55

Data were entered by two independent investigators and analyzed using SPSS 20.0 statistical software (SPSS Inc. Munich) and SAS 9.1. We received a certificate after an independent audit of the validity of the database by RPS (Clinical research organization; ReSearch Pharmaceutical Services, Nuremberg, Germany).

Sample size / power calculation
Sample sizes of at least 150 patients and 100 controls lead to a power in excess of 80% at the usual two-tailed significance level of 5% assuming a standardized mean difference of at least 0.37, i.e. at least a small to medium effect. For example, a standardized mean difference of 0.37 translates into a mean difference of 0.015 with a standard deviation of 0.04 for TCS in PD patients.49 These sample sizes result in a power of 90% to demonstrate that the AUC is larger than 0.7 given the true AUC is 0.8,56 which is a conservative assumption given that a previous ROC analysis of smell identification demonstrated an AUC of 0.91.57

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Table-e1: Assessment of non-motor features, neuropsychological and psychiatric symptoms

Non-motor features assessment (self-rating)

Assessment of autonomic dysfunction in Parkinson’s disease (Scopa-AUT)6, Parkinson’s Disease Questionnaire (PDQ-39)7, Non-motor Symptoms Questionnaire and –Scale (NMSQuest and NMSS)8-10
Sleep related symptoms (self-rating)

Parkinson’s Disease Sleep Scale version 2 (PDSS-2)11, Epworth Sleepiness Scale (ESS)12, 13, Medical Outcomes Study Sleep Scale (MOS-Sleep)*14, REM Sleep Behavior disorder Screening Questionnaire (RBD-SQ)15, REM-Sleep-Behavior Disorder Severity Scale (RBD-SS)16
Psychological assessment (self-rating and interview)
Psychiatric Symptoms
Beck Depression Inventory (BDI)28, Geriatric depression Scale (GDS)29, Montgomery Asberg Depression Rating Scale (MADRS)30, Apathy Evaluation Scale (AES) patient version and partner interview31, Neuropsychiatric Inventory (NPI)32, North-East Visual Hallucinations Interview (NEVHI)58
Neuropsychological assessment
Cognitive screening
Mini-Mental-Status-Examination (MMSE)18, Clock Drawing Test19, Montreal Cognitive Assesment (MoCA)59
Executive functions,
attention and speech
Similarities20
Verbal fluency21
Stroop test22
Wisconsin Card Sorting Test (WCST)60
Trail Making test (TMT)23
Memory
Verbal learning test (VLMT)24, Wechsler Memory Scale (WMS-R)25, Block Tapping Test26
Visuospatial function
Cube analysis, fragmented letters (VOSP)27
* only at baseline included

Table-e2: Results of the genetic analyses

Gene
Patients (n=159)
Controls (n=110)
Remark
Parkin (MLPA)
6 heterozygous exon deletions or duplications (3.8%)
Not tested
Slightly higher than the frequency in healthy German controls (3.2%)61, and real frequency in the DeNoPa patients may be higher since sequence alterations were only tested for in the 6 individuals carrying heterozygous gene dosage changes in Parkin
PINK1 (MLPA)
No mutation found
Not tested
None
DJ-1 (MLPA)
No mutation found
Not tested
None
SNCA
(MLPA, incl. A30P)
No mutation found
Not tested
None
LRRK2
(MLPA incl. G2019S)
No mutation found
Not tested
None
GBA (Ex8-11 seq)
3 heterozygous mutations (2x N370S;L444P; 2.5%)
None
GBA mutations have been reported as risk factor for PD; Frequencies fit with published data in non-Ashkenazi Jewish Europeans62
MAPT (allele count)
257x H1 allele (80.8%)
176x H1 allele (79.3%)
H1 allele has been reported as risk factor for PD; Frequencies fit with published data in Europeans63
SNCA – rs11931074
24x T allele
(7.5%)
12x T allele
(5.4%)
T allele has been reported as risk factor for PD; Frequencies fit with published data in Europeans63
SNCA – REP1
89x 259bp allele (28.0%)

18x 263bp allele (5.7%)
56x 259bp allele (25.0%)

11x 263bp allele
(4.9%)
259bp allele has been reported as being protective / 263bp allele as risk allele for PD

Table e-3: Performance of individual questionnaires and tests including area under the receiver-operating curve (ROC) (AUC) including the confidence interval (CI), optimal cut-off-values as determined by Youden Index64 and sensitivity preferred strategy54 and sensitivity and specificity.

Maximizing Youden Index
Sensitivity preferred strategy with sensitivity of at least 85%

AUC
(95% CI)
Cut-off
value
Sensitivity
Spe
cificity
Cut-off value
Sensitivity
Specificity
NMSQuest
0.748
(0.690 – 0.806)
5.7
0.67
0.78
2.9
0.89
0.35
Scopa-AUT gastrointestinal
0.723
(0.660 – 0.786)
2.0
0.67
0.84
1.0
0.85
0.59
Smell identifiation test
0.836
(0.785 – 0.886)
10.0
0.82
0.81
11.0
0.88
0.70
ECG (heart rate)
0.692
(0.629-0.755)
67.0

0.55
0.75
52
0.85
0.36
Serum cholesterol
0.633
(0.566-0.700)
251.5
0.85

0.36
281.5
0.85
0.36
TCS
(hyperechogenic substantia nigra)
0.897
(0.854 -0.940)
0.22
0.87
0.85
0.22
0.87
0.85
PSG*

0.51
(0.43-0.59)
0.85
(0.78-0.91)

NMS-Q: Non-motor Symptoms Questionnaire; Scopa-AUT: Assessment of autonomic dysfunction in Parkinson’s disease; TCS: transcranial sonography; PSG: Polysomnography
*For binary variable a ROC analysis is meaningless. Therefore only sensitivity and specificity are reported.

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