Biomarkers for Epilepsy Development and the Effect of Physical Exercise in Children With Autism

Blood sample collection for genetic and protein analyses

Participants will provide venous blood samples (maximum 10 mL per occasion, up to 2 occasions) for genetic analyses, focusing on synaptic proteins and circadian clock genes (exome sequencing of approximately 600 genes), as well as protein analyses using ELISA, Western blot, or equivalent techniques to assess immunologically associated proteins, including pro- and anti-inflammatory proteins and cell death-related proteins. Biological parents of the children will also provide blood samples for comparison in the genetic analyses. Parental consent for this blood collection will be obtained using a separate consent form.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Actigraphy to evaluate sleep-wake patterns: Sleep latency (minutes)

Sleep-wake patterns were evaluated using actigraphy. The children were asked to wear an actigraphy watch (MotionWatch 8, CamNtech, UK), which monitors movement and light exposure, for 5-7 days. Sleep latency (minutes) was measured as the duration between the time point the child tries to go to sleep (lights out) and the start of the first block of immobility (presumed fell asleep).

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Actigraphy to evaluate sleep-wake patterns: Assumed sleep (hours)

Actigraphy to evaluate sleep-wake patterns Description: Sleep-wake patterns were evaluated using actigraphy. The children were asked to wear an actigraphy watch (MotionWatch 8, CamNtech, UK), which monitors movement and light exposure, for 5-7 days. Assumed sleep (the sleep period; hours) represents the duration from fell asleep to the end of the last part/block of immobility before getting out of bed (woke up).

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Actigraphy to evaluate sleep-wake patterns: Fragmentation Index (index units)

Sleep-wake patterns were evaluated using actigraphy. The children were asked to wear an actigraphy watch (MotionWatch 8, CamNtech, UK), which monitors movement and light exposure, for 5-7 days. Fragmentation Index (index units) is used to measure sleep quality, composed of the percentage of measured mobile time and immobile bouts with a duration ≤1 min, during assumed sleep.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Actigraphy to evaluate sleep-wake patterns: Wake Bouts (seconds)

Sleep-wake patterns were evaluated using actigraphy. The children were asked to wear an actigraphy watch (MotionWatch 8, CamNtech, UK), which monitors movement and light exposure, for 5-7 days. Wake bouts (seconds) means single episodes of wakefulness during the sleep period. Total daytime activity score (score units) was defined by the sum of the activity counts during wakefulness..

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Actigraphy to evaluate sleep-wake patterns: Total Activity Score (score units)

Actigraphy to evaluate sleep-wake patterns: Wake Bouts (seconds) Description: Sleep-wake patterns were evaluated using actigraphy. The children were asked to wear an actigraphy watch (MotionWatch 8, CamNtech, UK), which monitors movement and light exposure, for 5-7 days. Total daytime activity score (score units) was defined by the sum of the activity counts during wakefulness.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Autism Spectrum Screening Questionnaire (ASSQ)

ASSQ is a screening questionnaire for autism spectrum disorder, containing 27 questions or statements assessing behaviors related to ASD. The ratings available are "no" (0 point), "somewhat" (1 point), "yes" (2 points). The points are added up to a total score of maximum 54 points. For a clinical population the score 19 are used as a cutoff to indicate autism spectrum disorder.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Behavior Rating Inventory of Executive Function , Second Edition (BRIEF-2)

BRIEF measuring executive functions and self-regulation in children and adolescents ages 5-18 in the parent form. The questionnaire consists of 63 statements and nine clinical scales: inhibition, self-monitor, shift, emotional control, initiate, working memory, plan/organize, task-monitor and organizing of materials. These scales are distributed under three domains: behavioral index, emotional index and cognitive index. The global executive composite (GEC) averages all assessment values. The rater records their answer via a Likert-type format N (never), S (sometimes) or O (often). Results are presented in T-score format (M=50, SD=10) and a higher T-score suggests that the child exhibits significant difficulties with executive functions. The results could also be compared to age-matched norms. Cut-off scores: 60-64 mildly elevated; 65-69 potentially clinically elevated; ≥70 clinically elevated difficulties.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Child Sensory Profile (CSP)

CSP is an assessment to evaluate a child's sensory processing patterns in different environments and how they impact on daily functioning. The parent form can be used for children ages 3-14 years. It consists of 86 statements and the parent record their answer by choosing between "almost always", "frequently", "half of the time", "occasionally", "almost never" or "does not apply". Most of the items are counted under one of the quadrants: seeking (the degree to which a child obtains sensory input), avoiding (the degree to which a child is bothered by sensory input), sensitivity (the degree to which a child detects sensory input) and registration (the degree on which a child misses sensory input). The form also provides information about how the child processes different modalities of sensory inputs divided into the following sensory sections: auditory, visual, touch, movement, body position and oral. Behavioral responses related to sensory inputs are divided into the following behavior

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Social Responsiveness Scale (SRS)

SRS aims to identify social impairment associated with ASD and quantify its severity. The school age form applies for parents to children and adolescents ages 4-18 years. It consists of 65 items which are rated on a graduated response scale: "never true", "sometimes true", "often true" and "almost always true". Five subscales are used for evaluating treatment: social awareness, social cognition, social communication, social motivation and restricted interest and repetitive behavior. The subscales are preferably not used for screening or diagnosis. The total and subscale scores are compared to clinical cut-offs indicating presence of autism-related social impairments; 60-75 mild to moderate social impairment; ≥76 severe social impairment.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Strengths and Difficulties Questionnaire (SDQ)

SDQ is an emotional and behavioral screening questionnaire for parents to children and adolescents ages 4-17 years. It consists of 33 questions or statements and the responder records their answer for the first 25 items on the response scale: "not true", "somewhat true" or "certainly true". The remaining items address the severity and the duration of the difficulties. The first 25 items asses five scales: emotional symptoms, conduct problems, hyperactivity/inattention, peer relationship problems and prosocial behavior. The total score helps to categorize the child or adolescent as having "normal", "borderline" or "abnormal" difficulties. For the subscales there are clinical cut-off scores.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline

Swanson, Nolan, and Pelham 26-item Rating Scale (SNAP-IV)

SNAP-IV is a 30-item rating scale for parents to assess symptoms of ADHD (inattention and hyperactivity/impulsivity) and symptoms of oppositional defiant disorder, based on the DSM criteria. It is divided into subscales for inattention, hyperactivity/impulsivity, and oppositional behavior. Symptom severity is rated on a 4-point scale: not at all (0), just a little (1), quite a bit (2) and very much (3). Subscale scores are calculated and compared to clinical cut-off scores. ≥ 1.67 indicates clinical difficulties for combinated inattention + hyperactivity/impulsivity.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Cognitive testing with CANTAB: Intra-Extra Dimensional Set Shift (IED)

For cognitive assessment, all children underwent a selection of tests from the computerized Cambridge Neuropsychological Test Automated Battery (CANTAB). IED includes 27 outcome measures to evaluate rule acquisition and reversal, requiring visual discrimination, attentional set formation, maintenance, shifting, and attention flexibility. The participant must work out a rule that determines which stimulus is correct. After six correct responses, the stimuli and/or rule changes. Outcome measure assess the number of stages completed, the numbers of trials completed, the number of errors made and response latency. Outcome measurements as they are described in CANTAB Connect Research: Admin Application User Guide, Cambridge Cognition limited 2022, V1.25. Note that each test has several more outcome measurements that we excluded in our analysis.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Cognitive testing with CANTAB: Paired Association Learning (PAL)

For cognitive assessment, all children underwent a selection of tests from the computerized Cambridge Neuropsychological Test Automated Battery (CANTAB). With 21 outcome measures, the PAL test evaluates visual memory and new learning abilities. Boxes are displayed and one or more of them contains a pattern. The patterns are then displayed in the middle and the participant must select the box containing the same pattern. Outcome measurements are stages completed, memory scores, the errors made, and the number of trials required to locate the patterns.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Cognitive testing with CANTAB: Reaction Time (RTI)

Comprising 21 outcome measures, this test assesses motor and mental response speed, as well as movement time, reaction time, response accuracy, and impulsivity. The participant must select and hold a button while circles are presented on the screen. When a circle with a yellow dot in it appears, the participant must react as fast as possible and select the circle with the dot in it. Outcome measures are reaction time and movement time.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Cognitive testing with CANTAB: Stockings of Cambridge (SOC)

Featuring 27 outcome measures, the SOC test assesses spatial planning and problem-solving strategy formulation. The participant is requested to move balls to copy a pattern and use as few moves as possible. Movement time is discounted in a part of the task where the participants just copy the movements made by the computer which mimics the movements the participant made when solving the original problem. Outcome measures include difficulty level reached, mean moves used and thinking time.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Cognitive testing with CANTAB: Spatial Working Memory (SWM)

The SWM test, with 24 outcome measures, assesses the retention and manipulation of visuospatial information, providing measures of strategy use and working memory. The participant is requested to select the boxes and using a process of elimination to find a token in each of a number of boxes. The number of boxes gradually increases, and the color and position of the boxes changes between trials. Outcome measures include different type of errors and strategy.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Evaluation of the intervention

Semi-structured or structured interviews with participants and/or parents.

Time frame: After completion of the intervention, within 6 months from baseline.

Sleep Electroencephalogram (EEG): Posterior Dominant Rhythm (Hz)

Participants will undergo a sleep EEG recording lasting approximately 40 minutes to assess brain electrical activity during sleep, to measure posterior Dominant Rhythm in Hertz (Hz), which is the main background alpha frequency.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Sleep Electroencephalogram (EEG): Interictal epileptiform activity (IEA)

Participants will undergo a sleep EEG recording lasting approximately 40 minutes to assess brain electrical activity during sleep, to measure IEA; the frequency of interictal epileptiform activity was assessed and graded as rare, moderate, or frequent.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.

Sleep Electroencephalogram (EEG): Sleep Spindles (counts)

Participants will undergo a sleep EEG recording lasting approximately 40 minutes to assess brain electrical activity during sleep, to measure sleep spindles; number of sleep spindles during the first 5 minutes following the first vertex wave, as well as after the occurrence of the first sleep spindle after sleep onset.

Time frame: Will be conducted at baseline (time of enrollment) and again 1-3 months after completion of the physical exercise intervention, within 6 months from baseline.