Early Biomarkers in Premanifest Huntington's Disease Gene Carriers: a Pilot Study

Huntington\'s disease (HD) is an autosomal dominant, neurodegenerative, fatal disease caused by a mutation in the hungtingtin (HTT) gene,1 manifesting with neurological, psychiatric and cognitive disorders. The disease causes a severe physical, functional and social impairment, with a great impact on patients' and their care-givers' quality of life. To date, the pathophysiology of HD remains largely unknown. Neuronal degeneration of basal ganglia plays a primary role in causing neuropsychiatric symptoms. Nevertheless, a cortical involvement has been evoked in the genesis of other symptoms of the disease, such as visual system disorders.2 To date only symptomatic treatment is available for motor and psychiatric issues in HD patients. The research field is now focused on investigation of genetic neuroprotective strategies to prevent the progression of the disease.3 The ideal therapeutic goal would be to start the treatment as soon as the disease becomes symptomatic, as the HD mutation is present since the birth. Unfortunately, the age and the symptoms at onset are very variable, mainly related to the mutation size4 but not only. Several other genetic and environmental factors are thought to play a role in the transition from the pre-symptomatic to the symptomatic stage, which remains largely unpredictable.5 Moreover, the diagnosis of symptomatic HD is traditionally based on motor symptoms onset (chorea, dystonia, bradykinesia, coordination issues).6 There are some evidence that subtle structural brain MRI alterations, mild cognitive and psychiatric issues may be present even in the pre-symptomatic stage, suggesting that the symptomatic phase could start before the onset of classical motor symptoms.7 Clinical studies using morphological and functional MRI have recently shown an early involvement of the visual cortex (area visual associative, lateral occipital cortex), which may explain the visuospatial function and visual perception disorders already observed at the pre-symptomatic and initial symptomatic stage of MH.8 The superior colliculus (SC) is a mesencephalic structure involved in the integration of visual stimuli, in the generation of oriented ocular saccades and attention tasks.9 An early dysfunction of SC has been shown in other diseases of the basal ganglia, such as Parkinson\'s disease (PD)10 and focal dystonia.11 This structure may also be early involved in the pathogenesis of visual disturbances and ocular saccadic changes described in HD.12 To date, there is no data regarding the functional state of SC in response to visual stimuli in HD.

Gait and balance issues in HD are common at the symptomatic stage of the disease, being a major risk of falls along with abnormal involuntary movements.13 They can be present at the early stage of the disease, with a mild severity. To date, no data are available about specific gait assessment in the pre-symptomatic HD carriers. Subtle gait issues resulting in spatiotemporal parameters alteration, not detectable with the standard clinical examination, could be present even in HD asymptomatic gene carriers. New technologies based on 3D virtual reality tools could be of great interest in the diagnosis of very early gait issues in pre-symptomatic HD subjects.

Cognitive issues are also common in HD, ranging from mild cognitive impairment in pre-manifest subjects14 to dementia in the advanced disease.15 Cognitive and emotional impairment is related to the basal ganglia degeneration in HD, progressively involving frontal and subcortical limbic regions.16 Eye-tracking data are known to be good markers of the orientation of attention. Recently, our team has shown that PD is associated in particular with dysfunction of the SC.10,17 At the same time, PD patients have difficulty producing and recognizing facial and vocal emotional expressions and are less reactive to negative stimuli than HS.18 As PD and HD share similar basal ganglia network dysfunctions, they could share also similar cognitive and emotional changes, which may be present from the onset of HD, due to structural and/or functional deficits in frontal and subcortical limbic areas. To date, no study has investigated emotional behavior and orientation reactions correlated with specific functional MRI (fMRI) protocol in pre-symptomatic HD carriers.

Objectives :

The primary objective of our study is to investigate the function of SC in response to visual stimulation using a fMRI in premanifest HD gene carriers (PMGC) compared to healthy subjects (HS). Our main hypothesis is the dysfunction of the SC at the pre-symptomatic stage of the disease (role of SC as an early biomarker of the disease).

Secondary objectives will be:

1. To study gait parameters using 3D virtual reality equipment in PMGC compared to HC, to demonstrate that they are abnormal at the pre-symptomatic stage of the disease. 2. To investigate the cognitive/emotional behavior in PMGC compared to HC, to demonstrate that it is abnormal at the pre-symptomatic stage of the disease. 3. To study the correlation between gait, neuroimaging, cognitive/emotional data and various clinical and neuropsychological variables. 4. To investigate potential pre-symptomatic subtypes profiles in HD carriers, based on neuroimaging, clinical, cognitive/emotional assessments correlation analysis.

Methodology :

Our study is a prospective, single-center, case-control, pilot study. Ten PMGC and ten age (+/- 5 years)- and sex-matched HS will be included. Main inclusion criteria will be: 1. For PMGC: HD diagnosis confirmed by genetic testing (CAG triplets ≥36 in HTT gene); Unified Huntington Disease Rating Scale19 Total Motor Score (UHDRS-TMS) ≤ 5; age 18-70 years old. 2. For HC: normal neurological examination; age 18-70 years old. Main exclusion criteria for both PMGC and HS will be: major ophthalmologic and psychiatric active diseases; cognitive impairment (Montreal Cognitive Assessment, MoCA score \< 25/30); gait issues related to non-neurological conditions; contraindications to brain MRI.

Study design:

Selection of study population and inclusion in the study (V0). The PMGC will be selected in the Movement Disorders Unit of Grenoble University Hospital during a neuro-genetic consultation after genetic testing performed at the Service of Genetics at CHU Grenoble Alpes. HS will be recruited by means of advertisements published on the CNRS cognitive science website (http://www.risc.cnrs.fr/), and posted in the Service of Neurology, research laboratories belonging to UGA, and on the HD patients association website (https://huntington.fr). Informed consent to the study will be signed by the participants. Inclusion and exclusion criteria will be verified and the neurological assessment will be realized by an experienced neurologist in movement disorders.

Ophthalmologic visit (V1).The PMGC and the HS will perform an ophthalmological examination (measurement of visual acuity, fundus of the eye, visual field). The maximal time interval between V0 and V1 will be 1 month.

Neuropsychological assessment (V2). A complete neuropsychological assessment will be performed in PMGC by an experienced neuropsychologist in movement disorders, including: a. assessment of executive functions using frontal score, Stroop Delis-Kaplan, letter/number sequence (oral version of TMT); b. Memory assessment using WAIS IV, Corsi blocks, RL/R116 and Doors test; c. Assessment of instrumental functions with the BJLO-15 items, VOSP, BECS; d. Behavioural assessment with the BDI-II, LARS, SHAPS, STAI, UHDRS (psycho-behavioural part).

Functional MRI session (V3). Pre-symptomatic subjects and HS will perform an fMRI session. The time interval between V2 and V3 will be set between 2 weeks and 2 months. The fMRI acquisitions will be performed during 1 hour under visual stimulation, according to the protocol developed by our team.

Gait assessment (V4). The gait parameters of PGMC and HS will be assessed during 1 hour-visit using a special 3D equipment in the Movement Disorders Unit. The time interval between V3 and V4 will be set between 2 weeks and 2 months.

Cognitive/emotional assessment (V5). The study population will perform two different experimental sessions: one using eye-tracking for a task involving free exploration of emotional stimuli and the other using fMRI coupled with eye-tracking for tasks based on emotional feelings and the tendency to act. The time interval between V4 and V5 will be set between 2 weeks and 2 months.

After the V5, the study will end. Neuroimaging assessment The neuroimaging procedure includes: anatomical and perfusion brain MRI, and fMRI with a visual stimulation protocol.8 fMRI procedure. MRI acquisitions will be performed at the IRMaGE MRI facility in Grenoble using a whole-body 3-Tesla Philips Achieva MRI scanner equipped with a 32 channel SENSE head coil. Participants will be instructed to fixate a cross localized at the center of a screen in front of their eyes, on which the visual stimuli is presented to control ocular saccades. During each functional session, PRL and eye movements will be monitored using the ASL 6000 eye-tracker. To maintain and control the attention, each participant will be instructed to press a button when the orientation of the cross changes. The visual stimulation protocol comprises a series of achromatic checkerboards with four levels of luminance contrast (1%, 3%, 5%, and 9%) and flashing at a frequency of 4Hz on a grey background. Each fMRI run is composed of four 12-s blocks of visual stimulation, one for each contrast (Figure 1), and five 12-s fixation intervals. The complete functional MRI session is composed of nine scanning runs, leading to 36 randomized block-presentations of each luminance contrast level. The cardiac signal is indirectly recorded based on the hemodynamic pulse at fingertip using the pulse plethysmography unit attached to the MRI scanner.

MRI sequences. Functional, anatomical, and perfusion measurements will be performed during each MRI session. For functional scans, a standard gradient echo planar imaging sequence will be used. Twenty-five slices (1.5 mm) will be acquired covering the SC, the lateral geniculate nucleus (LGN) and the primary visual area cortex (V1). High-resolution structural images will be acquired using T1-weighted 3D MP-RAGE and FGATIR sequences and used for manual delineation of our regions-of-interest (ROI) (Figure 2). A whole-brain pseudo-continuous arterial spin labeling (ASL) sequence will be used to measure brain tissue perfusion.

Gait assessment

The gait protocol assessment will be performed using a 3D specific equipment developed by Social Dreams® enterprise. This equipment is already available on the market, with CE authorization (www.socialdreams.fr). It has been applied in ongoing clinical studies investigating gait function and cognitive stimulation in patients with Alzheimer's disease in the Geriatric Service of the CHU of Lyon, France. Data not yet published). The equipment includes:

1. PERCEPTION NEURON3 (PN3, figure 3): a body multisensor-motion capture-integrated suite, able to capture any body movement, with no constraints on space, lighting or environment. The PN3 " Body Kit " includes: PN3 Body Sensors (17); PN3 Spare Sensor (1); PN3 USB Transceiver (1); Body Straps (1 set); Charging Docks (3); Axis Studio License dongle (1); USB-A to USB-C cable (3); USB-A Male to USB-C Female; Axis Neuron capture and recording software. 2. PICOG3 (figure 4): a 3DOF VR headset with 4K screen and integrated audio system, with six degrees of freedom, able to track the movements of both head and body, and to reproduce them in Virtual Reality with realistic accuracy, with no external sensors required. The kit includes also: a TP-LINK TL-WR902AC Routeur sans fil Dual-Band Wi-Fi AC (AC430+ N300); a tactile Tablette Lenovo Tab P11 - 11\'\' 2K LCD (Processeur Qualcomm Snapdragon 662 8Coeurs, 4 Go de RAM, UFS 64 Go, Qualcomm Adreno 610 GPU,