Study design
The study has two arms reflecting two study aims: A cross-sectional study of patients diagnosed with CJD, who are carriers of E200K mutation in the PRNP gene (gCJD), and a longitudinal study of first-degree healthy relatives (HR) (both carriers and non-carriers of the E200K mutation in the PRNP gene) of patients diagnosed with gCJD (Appendix A).
At baseline, and at the end of every year, participants are invited for an “in-depth” visit, which includes a clinical evaluation, blood and urine collection, gait assessment, brain MRI, lumbar puncture (LP), and PSG. At 6 months from baseline, and then halfway through each year, participants are invited for a “brief” visit, which includes a clinical evaluation, short cognitive assessment, and blood and urine collection (Fig. 1).
After obtaining informed consent, participants undergo a physical and neurological evaluation performed by a certified neurologist. The participant’s medical and family history will be reviewed as well as his or her concomitant medications. An experienced clinical researcher will then perform the cognitive and gait evaluations and will help participants fill in several questionnaires. The participant is fitted with a gait home-monitoring device and will have direct contact with the study personnel. Participants will be encouraged to contact the investigators if any symptoms or medical issues emerge between visits. Participants in the longitudinal study will then be scheduled for LP, MRI, and PSG tests. These procedures will be performed on different days but all within 90 days (preferably within 30 days) from the clinical assessment and the provision of the biological samples.
Study population
Inclusion/ exclusion criteria
gCJD patients' inclusion criteria:
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Diagnosis of CJD at baseline/ screening.
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Age 18 years or older at baseline.
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Confirmation of E200K mutation or willingness to undergo genetic testing as part of the pre-screening for new patients.
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Ability to provide written informed consent under Good Clinical Practice (GCP), International Conference on Harmonization (ICH), and local regulations, or having a person with a power of attorney who is willing to provide written informed consent.
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Willingness and ability to comply with scheduled visits, required study procedures, and laboratory tests.
gCJD patients' exclusion criteria:
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Any other medical or psychiatric condition or laboratory abnormality, which in the opinion of the investigator might preclude participation.
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Previously obtained MRI scan with evidence of clinically significant neurological disorder other than CJD.
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Current anticoagulant treatment (e.g Non-vitamin K Antagonist Oral Anticoagulants (NOACs), Warfarin, Low Molecular weight Heparin) that might preclude safe completion of LP.
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Conditions that preclude the safe performance of LP, such as severe lumbar spinal disease, bleeding diathesis, or clinically significant coagulopathy or thrombocytopenia.
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Conditions that preclude the safe performance of MRI scannings such as subjects who have a pacemaker, aneurysm clips, artificial heart valves, ear implants, metal fragments or foreign objects in the eyes, skin, or body, or any other known contra-indication for MRI.
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Active malignant disease.
Healthy relatives (HR) inclusion criteria:
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First–degree relative of an E200K gCJD patient.
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Age 50 years or older at baseline.
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Willingness to undergo genetic testing.
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Ability to provide written informed consent under GCP, ICH, and local regulations.
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Willingness and ability to comply with scheduled visits, required study procedures, and laboratory tests.
Healthy relatives’ exclusion criteria include a clinical diagnosis of CJD. Otherwise, exclusion criteria are the same as those described for gCJD patients.
Recruitment
gCJD patients will be recruited from the CJD clinic or the neurological department at the Tel Aviv “Sourasky” Medical Center (TASMC). Patients who are willing to participate will receive genetic counseling before signing informed consent. They will then be genotyped. Once their genotype is known they will be invited to participate in the study. HR of those patients will be invited to participate in the study if they fulfill the inclusion criteria. All potential participants will be approached by a neurologist who will explain the study procedures, the purpose, potential risks, and benefits of this study and informed consent will be obtained.
Sample size calculation
Based on the Israel National Registry for CJD, the annual incidence rate of CJD is 51 cases, although this is believed to be an underestimation. About two-thirds of the reported cases are familial cases, and of these cases, almost all are carriers of the E200K mutation. Based on Spudich et al. (Spudich et al., 1995), the likelihood of developing gCJD is 0.45 in the age group of 51–60. According to Minikel et al, the average annual probability of onset for healthy carriers aged 40 to 80 years is 4.6% (Minikel et al., 2019). Using conservative power estimation, assuming approximately 3.8% per year pheno-conversion (13.5% for the whole study period) and 80% power, we will aim to include a total of 106 healthy first-degree relatives of patients with gCJD (53 healthy mutation carriers and 53 non-carriers), as well as 20 patients with gCJD.
Methods
Clinical neurological and cognitive evaluation:
Healthy relatives of CJD patients will undergo the following longitudinal assessments:
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Collection of clinical data, including cognitive, behavioral, and autonomic function.
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Collection of Blood, urine, and CSF biomarkers.
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Electrophysiological measurements and assessment of other sleep characteristics.
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structural and functional markers on brain MRI.
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Gait and movement assessments.
Data regarding demographics, medical and family history as well as physical and neurological examinations will be collected on all participants. Participants will be asked to provide information as to food and drink consumption on the day of the exam. Concomitant medications, including over-the-counter dietary supplements (e.g., herbal remedies), or prescription medications, will be recorded. Evaluation of individuals who take antibiotics will be deferred until their course of antibiotics ends, with a washout period of at least 1 week.
All gCJD patients will be scored according to the National Prion Monitoring Cohort (NPMC) Scale (MRC Prion Disease Rating Scale) (Thompson et al., 2013), which is a multimodal scale assessing both cognitive impairment and daily function disability. This scale consists of 11 parameters: bowel function, bladder function, toilet use, bathing, feeding, transfer and mobility, stairs, best verbal response, memory and orientation to surroundings, judgment and problem solving, and use of tools. Each section is scored between 0–4 with 0 indicating complete disability and 4 normal ability, for a maximum of 20 points.
Cognitive assessment
Standardized neuropsychological tests assessing different cognitive domains will provide an in-depth evaluation of cognitive function. These tests include the following:
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Montreal Cognitive Assessment (MoCA) (Rossetti et al., 2011) - A 30-point cognitive screening instrument that assesses visuospatial, executive, naming, attention, language, abstraction, delayed recall, and orientation domains.
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Verbal Fluency tests (Kavé and Knafo-Noam, 2015) on which participants name as many different words that begin with a designated letter or as many items that belong to a given semantic category in one minute. The score is the sum of all correct words produced.
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The Motor-free Visual Perception Test (MVPT-3) (Brown et al., 2010), which measures the ability to discriminate dominant features of different objects, including the ability to discriminate position, shapes, and forms, and the ability to perceive the positions of objects in relation to oneself and other objects. This test also examines visual memory, the ability to distinguish an object from the surrounding objects, and the ability to perceive a whole figure from its fragments.
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The Frontal Assessment Battery (FAB) (Dubois et al., 2000) targets executive functions, including attention span, working memory, motor planning, abstraction, and inhibition.
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Digit span (Wechsler Adult Intelligence Scale, Third Edition, WAIS-III) which measures verbal working memory by asking participants to repeat digits forward and backward.
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The Cookie theft picture description from the Boston Diagnostic Aphasia Examination–Third Edition (BDAE).
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Trail Making Test (TMT) parts A & B (Smith et al., 2008) which estimates visual search, speed of processing, and shifting attention.
Affect, behavior, and habits
The following questionnaires assess the level of anxiety, depression, stress, and personality traits:
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The Beck Depression Inventory (BDI) (Beck et al., 1961) which evaluates the presence and intensity of depression.
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The State-Trait Anxiety Inventory (STAI) (Spielberger, 1971) which measures emotional state anxiety, using 40 items.
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The Social Readjustment Rating Scale (SRRS) (Gerst et al., 1978) aims to identify major stressful life events.
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The Big-5 Questionnaire (Goldberg, 2001) measures dimensions of personality through ratings of 44 items.
Autonomic function
Questionnaires assessing autonomic function and sleep will be completed:
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The Epworth Sleepiness Scale (ESS), a self-administered questionnaire collecting information on the propensity to fall asleep in eight different situations encountered commonly in daily life. Each situation is rated from 0 (no chance of dozing) to 3 (high probability of dozing), and the total score ranges from 0 to 24. Total scores of zero to 10 are normal, 10–12 are borderline, and scores from 12 to 24 are abnormal.
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The REM Sleep Behavior Disorder Screening Questionnaire (RBDSQ) is a 10-item self-rated questionnaire to assess sleep-wake disturbances. Sleep behavior disorder may represent early manifestations of progressive neurodegenerative disorders, including Parkinson's disease. A score greater than 5 reflects RBD.
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SCOPA-AUT (Scales for Outcomes in Parkinson's disease - Autonomic Dysfunction) - The SCOPA-AUT was developed to evaluate autonomic symptoms in patients with Parkinson’s disease as well as patients with Multiple System Atrophy. The scale is self-completed by patients and consists of 25 items assessing the following domains: gastrointestinal (7 items), urinary (6 items), cardiovascular (3 items), thermoregulatory (4 items), pupillomotor (1 item), and sexual (2 items for men and 2 items for women).
Blood and urine samples
Samples handling and pre-processing
Participants will provide blood and urine samples when clinically assessed. They will be strongly encouraged to eat a low-lipid diet on the day of the test. Information on food intake will be collected. Samples will be delivered to the laboratory as soon as possible, and not more than 30 minutes after collection. Processing and extractions will occur immediately after the arrival of samples in the laboratory.
Serum extraction: To ensure serum clotting, the serum tube will be centrifuged at least 30 minutes after blood drawing (and no longer than 60 minutes) at 1500g for 15 minutes at 4°c. Then, on ice, the supernatant will be transferred to a conical 15ml tube and then inverted gently. The supernatant will be evenly divided into four aliquots and frozen at -80°c.
Plasma extraction (K2EDTA tube): Tubes will be centrifuged for no longer than 30 minutes after blood drawing at 2000g for 15 minutes at 25°c. The supernatant will be transferred to a conical 15ml tube and then inverted gently. Plasma will be divided into 8 aliquots of 0.5ml and frozen at -80°c. Buffy Coat (BC, K2EDTA tube): Immediately after transferring the plasma from the K2EDTA tube, BC will be cautiously collected to a labeled cryotube at RT and frozen at -80°c.
Urine processing: Urine tubes will be centrifuged upon arrival at 2500g for 15 minutes at 4°c. Then, on ice, the supernatant will be transferred to a conical 15ml tube and then inverted gently. The urine will be divided between two conical 15ml tubes, 4ml to the first tube and the remainder to the second. If the total amount of urine is less than 5ml, the urine will be divided evenly between the tubes. The tubes will be frozen at -80°c.
Whole blood tubes will be frozen no longer than 60 minutes after the blood draw.
PAX tubes will be collected for future RNA extraction. The tubes will be kept at room temperature for 3–26 hours and then frozen at -80°c. Whole blood for DNA extraction will be collected only at the first visit. The tube will be placed at 4°c until DNA is extracted.
Sample data will be inserted into a dedicated database software.
Genetic testing
Genomic DNA will be extracted from whole blood samples and will be genotyped for the PRNP-E200K mutation (rs28933385, TaqMan genotyping assay C_27531205_10_F_; Applied Biosystems, Foster City, CA, USA). Confirmation for the PRNP mutation will be done by a polymerase chain reaction and Sanger Sequencing. Confirmation by sequencing will be performed for healthy relatives that are shown to be E200K carriers and for all CJD patients.
Clinical laboratory tests
Blood will be collected for standard clinical laboratory tests: Complete Blood Count, Chemistry screen, Glycohemoglobin test, Vitamin D, Vitamin B12, Thyroid Function Tests, C-reactive protein, Lipid profile, International Normalized Ratio, Prothrombin Time, and Partial Thromboplastin Time.
Other fluid biomarkers in blood will include total-tau (T-tau), phosphorylated-tau (P-tau), Neurofilament light chain (NfL), Glial fibrillary acidic protein (GFAP), and Ubiquitin carboxy-terminal hydrolase L1 (UCHL-1).
Lumbar puncture (LP)
LP will be performed by an experienced neurologist at the Neurological Institute at TASMC. An LP for the collection of 15–20 ml of CSF will be carried out on all participants in the longitudinal study at baseline and at the end of every year thereafter, and when phenoconversion is suspected unless there is evidence of clinically significant coagulopathy or thrombocytopenia that would interfere with the safety of the procedure. LP will be performed with an a-traumatic needle (G25 pencil LP, AVMAD). Participants will be monitored during the procedure and following the procedure and will remain under bed rest supervision in the unit for 1.5 hours following the procedure. They will be contacted by phone the day after the procedure as well as 7 to 10 days following an LP to monitor any adverse events.
The first 2ml of CSF will be processed to conduct standard analyses on cell count, protein and glucose levels. The rest of the CSF will be centrifuged at 2000g for 10 minutes at 24°C and separated into 500ml aliquots in 1.5ml tubes, four of them with 0.03% CHAPS. The pellet will be suspended in 1.5ml of RNAlater Solution, placed at 4°C for approximately 24 hours, and stored at -80°C.
Other fluid biomarkers in CSF will include T-tau, P-tau, NfL, Neurogranin, Neuronal glycolytic enzyme (NSE), GFAP, YKL-40 (also known as Chitinase 3-like 1), UCHL-1, S100B, PrPsc, and Mitochondrial malate dehydrogenase 1 (MDH1) levels.
Imaging
Participants in the longitudinal study will undergo an MRI brain scan at the baseline visit, 12, 24, 36, and 48 months thereafter, and if phenoconversion is suspected. MRI scans will be performed at the Sagol Brain Institute, using a 3-T Magnetom Prisma (Siemens Healthineers, Erlangen, Germany) with a 20 channels phased-array head coil. MRI protocol will include structural imaging including T1 and T2-FLAIR weighted imaging to assess brain structure and pathology; T1, T2, and T2* relaxometry, to quantitatively assess the tissue relaxation times Diffusion Tensor Imaging (DTI) to assess tissue maturation and structural connectivity; resting-state fMRI – to assess functional connectivity. The imaging parameters of each sequence are given in Table 1.
Table 1
Sequence
|
TR ( Repetition time) [ms ± SD]
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TE (Echo time) [ms ± SD]
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In-Plan Resolution (XxY) [mm2]
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Slice Thickness [mm]
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Number of Slices
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Scanning time (min)
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T2-FLAIR
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8000
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117
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0.8X0.8
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5
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32
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2.58
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T1-MP2TAGE
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5000
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3.43
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1X1
|
1
|
176
|
7.07
|
DTI+
|
7200
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55
|
1.8X1.8
|
1.8
|
76
|
7.57
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rsfMRI
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2500
|
30
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2.3X2.3
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3
|
42
|
6.08
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T2*
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1350
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++2.8–80 (8 TEs)
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0.9X0.9
|
3
|
32
|
6.18
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T1-MPRAGE
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2200
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3.22
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1X1
|
1
|
192
|
5.06
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T2-map
|
2670
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+++17.5–105 (6 TEs)
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0.6X0.6
|
3
|
24
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6.59
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++++T1-IR
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2500
|
12
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0.7X0.7
|
4
|
24
|
4.02
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+30 directions, with 3 b-values: 0, 500 and 1000 s/mm.
++TEs = 12.81, 11.68, 23.07, 34.46, 45.85, 57.24, 68.63, 80.00 ms
+++TEs = 17.5, 35.0, 52.5, 70.0, 87.5, 105.0 ms
++++With inversion time = 500 ms
PSG, EEG monitoring, and physiological measurements during sleep
PSG includes EEG monitoring combined with physiological measurements during wakefulness and throughout overnight sleep as follows. High-density EEG is recorded continuously using a 256-channel hydrocel geodesic sensor net (Magstim Electrical Geodesics, Inc. system [Magstim-EGI]). Each carbon-fiber electrode consists of a silver-chloride carbon fiber pellet, a lead wire, and a gold-plated pin, and is injected with conductive gel (Electro-Cap International). Signals are referenced to Cz, amplified via an AC-coupled high-input impedance amplifier (NetAmps 300, EGI), and digitized at 1000 Hz. Electrode impedance in all sensors is verified to be < 50 kΩ before starting the recording. EEG is band-pass filtered offline between 0.5 Hz and 45 Hz, and an additional notch filter at 50 Hz is applied to the continuous data offline to remove residual line noise.
Beyond EEG, additional physiological signals include synchronized video, electrooculogram (EOG), electromyogram (EMG), heart rate measurements via chest electrodes, breathing assessment including SPO2/saturation, nasal airflow, and belts monitoring chest and abdomen efforts.
Gait rhythmicity, arm swing, and axial rotation
Participants will be asked to perform motor tasks in the clinic while wearing lightweight accelerometers on their lower back and wrists. A total of 3 tasks will be administered:
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Sway: Participants will be asked to stand with their feet together for 30 seconds while their eyes are open and then for 30 seconds while their eyes are closed to assess sway and subtle changes in stability. Velocity, direction, jerk, and displacement of the center of mass during the task will be determined.
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Gait: Participants will be asked to walk for one minute under two different walking conditions: comfortable speed and walking while performing a cognitive task of serial subtraction of 7 from a predefined 3-digit number. Swing time, average stride time, stride and swing time variability, and magnitude and symmetry of arm swing and axial rotation will be determined.
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The Timed Up and Go (TUG): This performance-based measure evaluates sit-to-stand transfer, walking for 3 meters, turning, and coming back to sitting on a chair. The TUG will be performed twice.
These validated tests provide information on fall risk (time of performance), transfer abilities, turning, and ability to follow instructions.
Ambulation and activity at home and in the community will be assessed using a body-worn sensor. Mobility quantity and quality (e.g., number of steps taken over 7 days, gait quality) will be measured using a small, lightweight, waterproof body-fixed sensor (i.e., accelerometer) worn on the back for 7 consecutive days following the assessment in the clinic. The device (AX3, Axivity Ltd, UK) allows for objective determination of the amount and type of activity performed by the participant. This, for example, includes time spent lying, sitting, standing, and walking and the quality of each of these activities over every 7 days. These measures reflect different aspects of health status and are also related to disease outcomes. In addition, the quality of mobility will be assessed by evaluating step time, variability, and rhythmicity of movement. The device will be returned to the clinic after 7 days via courier service.