Participants
3 SYNE1, 6 Friedreich ataxia patients and 12 healthy controls were enrolled in the study. The patients underwent a detailed diagnostic approach including neurological examination, laboratory and radiological investigations to exclude acquired causes of ataxia. Scale for the Assessment and Rating of Ataxia (SARA) scores were recorded in all cases. After obtaining written, informed consent, genomic DNA was extracted from peripheral blood leukocytes by standard protocol. First, according to recent guidelines on the management of sporadic ataxias without known secondary etiology [20], the most common repeat expansion hereditary ataxias (spinocerebellar ataxia 1, 2, 3, 6, 7 and FA) were tested. These examinations reinforced the diagnosis of FA in six patients. All of them have homozygous GAA repeat expansions in the first intron of the FXN gene. In the additional three patients, targeted gene analysis followed by new generation sequencing were performed.
For proband AT-04, whole exome sequencing (WES) was performed with SureSelectXT Human kit All Exon v7 (Agilent, Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instruction and paired-end sequenced (2x100 bp) on HiSeq 1500 (Illumina, San Diego, CA, USA). Prioritized variants were validated in the proband, proband’s parents and brother by amplicon deep sequencing performed using Nextera XT Kit (Illumina) and sequenced on HiSeq 1500 (Illumina).
For subjects AT-05 and AT-06 a total of 60 ng of genomic DNA was used for library preparation and sequenced with Trusight One clinical exome kit (Illumina) on Illumina MiSeq platform. The clinical exome kit covers the coding region of 4813 clinically relevant, disease-associated genes. The 150 bp paired reads were aligned to the GRCh37.75 human reference genome by Burrows Wheel Aligner (BWA v0.7.9a) software. The variants were called by Genome Analysis Toolkit HaplotypeCaller (GATK v3.5) best practice; annotated by SnpEff and VariantStudio softwares. Variants were filtered based on severity and frequency against public variant databases including dbSNP, ClinVar, ExAC, EVS and an in-house clinical exome database of 140 unrelated Hungarian patients.
Eye tracking
Recording system
The used system and paradigm were described in a previous study [21]. The assessment was performed in a well-lit room. Subjects sat in front of the monitor and their head were fixed at 60 cm distance from the screen. We used a Tobii TX300 eye tracker and tasks were programmed in Psychophysich Toolbox V 3.0.12, under MatLab. Before every paradigm a five-points calibration was performed.
Saccade task
Subjects accomplished a visually guided saccade task as described here: a black cross appeared at the center of the screen and 1.2-2 seconds later it jumped to the right or left side of the screen. The background was grey and the distances of displacement of the cross were 9.2° or 18.4° horizontally. All measurements were repeated 20 times in a pseudorandom order, this means 80 measurements per subject. The participants had to shift their gaze to the new position of the target as fast and accurate as they can. There was a break in halftime of the task to prevent subjects tearing and/or tiring.
Antisaccade task
In the antisaccade task the composition was similar to the visually guided saccade paradigm, however, the participants had to direct their gaze to the opposite direction (e.g. if the target appeared in the left side, they had to look to the ride side). Only horizontal movements were recorded such as in the saccade task. There was also a pause in half of the trial.
Data acquisition and processing
Data recording began when the target jumped to the periphery and stayed there for one second. The recording frequency was 300 Hz and both eyes were registered separately. We used a semi-automatic in-house made script to define parameters of saccades as described in a previous study [21]. The following parameters were measured: peak velocity, latency, amplitude, gain and duration. In the saccade task, we assessed the main sequence relationships of duration versus amplitude and peak velocity versus amplitude using the linear model [22]. Additionally, in the antisaccade paradigm the incorrect ratio of antisaccades was also examined, which is a quotient showing the incorrectly executed antisaccades.
Neuropsychological assessment
The enrolled ataxia patients were assessed via cognitive examination performed by trained neuropsychologists. The global cognitive performance was measured by the Addenbrooke’s Cognitive Examination (ACE) including the Mini-Mental State Examination (MMSE). The executive function was evaluated by verbal and semantic fluency tests. In addition, the working memory and the ability of maintenance and manipulation of information were estimated by the Backward Digit Span Task (BDST) and the Listening Span Task (LST). The quality of information planning and visuoconstructional and visual organizational abilities were assessed by the Rey Complex Figure Test (RCFT).