Biomarker Research in ADHD: the Impact of Nutrition

Study Overview
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Brief Summary
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Attention deficit hyperactivity disorder (ADHD) is the most common childhood behavioural disorder, causing significant impediment to a child's development. The exact aetiology of ADHD is still unknown. It is a complex disorder with numerous contributing (epi)genetic and environmental factors. Currently, treatment predominantly consists of behavioural and pharmacological therapy. However, medication use is associated with several side effects and concerns about long-term effects and efficacy exist. Therefore, there is considerable interest in the development of alternative treatment options. Double-blind research investigating the effect of a few-foods diet (FFD) has demonstrated large improvements in ADHD symptoms. However, following an FFD requires great effort of both the child and parents. To make this treatment easier or potentially obsolete, it is important to understand how and in which children an FFD affects ADHD symptoms. The investigators hypothesise that an FFD affects brain function and behaviour, including ADHD symptoms, via the complex network of communication between the microbiota, gut and brain, i.e. the MGB axis. The aim of this study is to identify potential mechanism(s) underlying the impact of an FFD on ADHD symptoms and to identify biomarkers that predict the response to the FFD. 100 boys with ADHD will follow the FFD for 5 weeks. After inclusion, all participants will start with a baseline period, during which they will maintain their regular diet. The baseline period ends at the end of week 2. Thereafter, participants will follow a 5-week FFD, preceded by a 1-week transition period. The FFD period ends at the end of week 8. At the end of the baseline period (i.e. at the end of week 2) and at the end of the FFD (i.e. at the end of week 8), fMRI scans will be made, blood and buccal saliva will be collected, and stool and urine will be handed in. Children will do computer tasks and parents will complete questionnaires to monitor ADHD and physical complaints. All samples will be analysed by researchers blinded to behavioural responses to the FFD. To assess the impact of the FFD on brain function and the MGB axis, associations between ADHD behavioural changes and changes in other primary and secondary study outcomes will be analysed. This study may lead to the identification of biomarkers that can predict the response to an FFD. Understanding which changes - induced by an FFD - lead to improvements in ADHD symptoms may provide new avenues for developing treatments. Ultimately, the findings may enable personalised intervention strategies based on an individuals' configuration of the MGB axis.

Official Title
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Biomarker Research in ADHD: the Impact of Nutrition (BRAIN). An Open-label Trial to Investigate the Mechanisms Underlying the Effects of a Few-foods Diet on ADHD Symptoms in Children

Conditions
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Attention Deficit-Hyperactivity Disorder

Intervention / Treatment
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* Other: Few-foods diet


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Meeting DSM-IV ADHD criteria Male Aged 8 up to and including 10 years Right-handed Available to visit Wageningen University for 4 sessions (i.e. intake, screening, T1 and T2), of which 3 sessions including the child (screening, T1, T2) Upon study start, fully understanding and agreeing to the study objectives and having dated and signed an informed consent to participate in the study, including permission that material will be used or archived for (epi)genetic testing Willing to be informed about chance-findings that may have implications for the health of the child or his family, and approving of reporting this to the child's medical specialist or family's general physician. If the child uses over the counter medication, e.g. laxatives, melatonin for sleeping problems or hay fever medication, parents are asked to share the information leaflet, and if necessary participants are asked to change to alternatives that are free of additives that may affect ADHD, e.g. laxatives free of artificial sweeteners, sugar and cacao. Exclusion Criteria: Diagnosis Autism Spectrum Disorder Diagnosis Developmental Coordination Disorder Premature birth (< 36 weeks) and/or oxygen deprivation during birth Diagnosed chronic gastrointestinal disorder, i.e. inflammatory bowel disease, irritable bowel syndrome, celiac disease, non-celiac gluten-intolerance (gluten-sensitivity) or lactose-intolerance Auto-immune disorder (e.g. diabetes mellitus type 1) Vegetarian/vegan Diagnosis dyslexia and/or dyscalculia IQ < 85 Following behavioural therapy Use of ADHD medication Use of systemic antibiotics, antifungals, antivirals or antiparasitics in the past six months Insufficient command of the Dutch language by either parents or child that may affect understanding and execution of study and dietary instructions Family circumstances that may compromise following or completion of the diet, including but not limited to family relational problems Having a contra-indication to MRI scanning (including, but not limited to): pacemakers and defibrillators, intraorbital or intraocular metallic fragments, ferromagnetic implants, claustrophobia. Two weeks prior to the start of the study, dietary supplements (e.g. antioxidants, minerals, vitamins) or pro- or prebiotics use has to be stopped.

Ages Eligible for Study
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Minimum Age: 8 Years
Maximum Age: 10 Years

Sexes Eligible for Study
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Male

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Intervention Model: Single Group Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Few-foods diet intervention<br> | Other: Few-foods diet<br>* The few-foods diet (FFD) is followed for 5-weeks preceded by a 1-week transition period during which the child's eating pattern will be gradually adjusted. The diet consists of rice, meat (turkey and lamb), a range of vegetables, pear, rice milk with added calcium and water, and is complemented with foods such as potatoes, fruits, corn, some sweets and wheat, which are allowed in small quantities only. Normal quantities of vegetables, rice and meat are allowed every day. If necessary the diet will be adjusted to avoid foods that the child dislikes or has cravings for. If the child does not respond to the initial FFD, i.e. no change in behaviour after the first two weeks, interim adjustments to the FFD will be made in consultation with the parents.<br>* Other names: Restricted Elimination Diet;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in neural activation patterns during execution of tasks | Using fMRI, blood oxygen-level-dependent (BOLD) signal changes will be measured whilst performing cognitive tasks that assess inhibitory control and selective attention, i.e. a stop-signal task (response inhibition) and a Flanker task (response conflict and associated error monitoring). fMRI BOLD responses will be assessed between variable task-elements and performance. Region of interest (ROI; anatomically defined regions in the brain) analyses of the BOLD responses will be performed. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in peripheral blood metabolite concentrations | Global metabolite profiles will be examined in plasma (or alternatively serum) obtained from whole blood using mass-spectrometry profiling. Phenylalanine and tyrosine plasma levels represent primary outcomes. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in functional composition of the gut microbiota | Metagenome profiling will be performed on stool samples, leveraging Illumina next-generation sequencing technology. Sequence read data will be used for abundance profiling of microbiota genes that encode enzymes directly involved in the production or degradation of the dopamine and noradrenaline precursors phenylalanine and tyrosine. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in ADHD symptom scores | ADHD symptoms will be scored using the 18-item ADHD rating scale, which is based on the Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV and consists of 9 items that assess inattention and 9 items that focus on hyperactivity and impulsivity. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in whole brain neural activation patterns during the execution of tasks | fMRI BOLD responses, assessed between variable task-elements, will be explored using whole brain imaging analyses. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in whole brain functional connectivity at rest | A resting-state fMRI scan will be performed to analyse the networks of brain structures that are active during the resting-state. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in taxonomic and functional composition of the gut microbiota | Stool metagenome and/or 16S ribosomal ribonucleic acid (rRNA) gene profiling will be performed using Illumina sequencing. Metagenome data will be used to determine the taxonomic and functional composition of the gut microbiota. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in metabolite profiles | Global metabolite profiles will be examined in plasma (or alternatively serum) obtained from whole blood, urine and optionally in stool using mass-spectrometry profiling. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in peripheral blood cell gene expression profiles | Peripheral blood mononuclear cells will be isolated from fasting blood samples and gene expression profiles will either be determined using Affymetrix gene expression arrays or by RNA-sequencing. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in a panel of peripheral blood protein biomarkers related to immune, metabolic and neurological status | A large panel of proteins will be profiled using quantitative immunoassays or proteomics on blood plasma or serum. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| DNA genotype | Genotyping will be conducted using a microarray platform on DNA isolated from buccal cells or on DNA isolated from whole blood. | Before the FFD diet intervention (i.e. at the end of week 2) | 
| Change in DNA methylation profiles | Genome-wide profiling of DNA methylation status will be conducted using the Illumina Infinium MethylationEPIC beadchip microarray platform in DNA isolated from buccal cells or alternatively on DNA isolated from whole blood. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in executive function | Executive functioning will be measured using a continuous performance test that assesses executive functions such as sustained attention and behavioural inhibition. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in ADHD symptoms | Change will be scored using the Abbreviated Conners' scale | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in oppositional defiant disorder symptoms | Change will be scored using a validated questionnaire. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in social behavioural problems | Change will be scored using the children's social and behavioural questionnaire. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in physical complaints | Change will be scored using a validated questionnaire. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in stool frequency | The child will record the frequency of defaecations for one week. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 
| Change in stool type | The child will type each stool using the modified Bristol stool scale form for children, which comprises 5 stool form types described and depicted in drawings. | Before and at the end of the FFD intervention (i.e. at the end of week 2 and at the end of week 8) | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Attention Deficit Hyperactivity Disorder, ADHD, child, human, diet, few-foods diet, brain, gut, microbiota