Epigenetics in ADHD
Condition: microRNAs in Attention-deficit/Hyperactivity Disorder
MeSH terms: Hyperkinesis; Attention Deficit Disorder with Hyperactivity
Study type: Observational

Summary: 
      Attention-deficit/hyperactivity disorder (ADHD) is among the most common psychiatric
      disorders in children and adolescents. ADHD's nosology is largely based on clinical
      phenomenology that includes such symptoms as inattention, hyperactivity and impulsivity.
      However, a reliable ADHD biomarker has still not been determined either for differential
      diagnosis or for monitoring treatment effects. MicroRNAs (miRNAs) are small non-coding RNA
      molecules that function in the process of RNA silencing and the post-transcriptional
      regulation of gene expression. MiRNAs are found in abundance throughout the nervous system
      and play a vital role in the transcriptional networks with regards to human brain
      development. Currently, miRNAs' involvement in the pathogenesis of ADHD continues to be
      unclear. Therefore, this study aims to investigate the prospective role of miRNAs in ADHD and
      to determine whether miRNA levels in peripheral blood can serve as a biomarker and a
      diagnosis panel for ADHD.

      In the preliminary study, blood samples were collected from five patients with ADHD and five
      healthy control subjects. The use of Next Generation Sequencing (NGS) techniques has
      identified 23 miRNAs as potential biomarkers for ADHD. During this three-year proposal, we
      intend to recruit 100 drug-naïve patients with ADHD and 100 age- and gender-matched control
      subjects (Training Set). Blood will be obtained through direct puncture of the vein from each
      participant to analyze the miRNAs by using quantitative reverse transcription polymerase
      chain reaction (qRT-PCR). The behavior and neuropsychology of each participant will be
      assessed. This research will construct a miRNAs diagnosis panel using the Support Vector
      Machine (SVM) classification model to discriminate ADHD from non-ADHD. The validity of the
      miRNA diagnosis panel will then be re-examined using an independent validation sample
      composed of 50 patients with ADHD and 50 control subjects (Testing Set). All of the 150
      patients with ADHD will receive treatment in a traditional clinical practice and then will be
      followed up with for 12 months. At the twelfth month, the same procedures as those performed
      at the baseline will be replicated to examine the influence of ADHD medications on miRNAs, as
      well as determine whether miRNAs can serve as a biomarker to portray the condition of ADHD
      under treatment. MiRNA target gene prediction and functional annotation analysis will also be
      performed.

      This study will develop a potential diagnostic panel for ADHD through the use of combinations
      of multiple miRNA expressions. We will provide proof of the relationships of miRNAs profiles
      and ADHD manifestations in clinical samples and further explain the molecular pathogenesis of
      ADHD. Such information may become an important reference for future research and clinical
      treatments for patients with ADHD.
    

Detailed description: 
      Year 1: Recruiting 100 ADHD patients and 100 healthy control subjects (Training Set) to
      develop a miRNA diagnosis panel for ADHD

      1. One hundred eligible ADHD patients that are being treated in the Outpatient Department of
      Child Psychiatry at Kaohsiung Chang Gung Memorial Hospital in Taiwan will be recruited for
      this study. One hundred age- and gender-matched control subjects will be found in the same
      catchment area as that of the patients. After obtaining informed consent from their parents
      or guardians, research psychiatrists shall interview the participants using the K-SADS-E to
      confirm the diagnosis. The participants will also undergo WISC-IV assessment to exclude
      intellectual disabilities.

      2. For both the ADHD patients and the healthy control subjects, experienced child
      psychologists will administer Conners CPT and Conners CATA testing in a room designed to
      reduce inconsistent testing conditions. Parents or guardians will be required to complete the
      SNAP-IV parent form and the SAICA; patients' teachers will be required to fill out the
      SNAP-IV teacher form; and the ADHD-RS and the CGI will be completed and rated, respectively,
      by a child psychiatrist.

      3. Venous blood will be collected from each participant in an EDTA anticoagulant tube. The
      blood samples will be further processed with the mirVana miRNA isolation kit (Life
      technology) to extract total RNAs. Based on the preliminary findings, the ΔCt values of the
      22 candidate miRNAs will be determined for the 100 ADHD samples and 100 control samples. Then
      the 10 miRNAs with the smallest p-value will be chosen to develop a miRNAs diagnosis panel
      for ADHD in the second year.

      4. The ADHD patients and healthy control subjects will have 12 months of follow-up. The
      treatment procedures during that time for patients with ADHD will be determined by the
      patients' psychiatrists' traditional practice and the agreement of patients and their
      parents.

        1. ADHD patients who decide to receive medical treatment will be prescribed oral
           short-acting MPH (MPH-IR) two or three times daily, with each dose ranging between 0.3
           and 1.0 mg/kg, which shall be determined based on the severity of their clinical
           symptoms, as well as their age, height, and body weight.

        2. The dosage will be adjusted every two weeks to properly improve ADHD symptoms. Changes
           to long-acting MPH (Ritalin-LA) or extended-release methylphenidate (Concerta) are
           permitted depending on clinical requirements. The maximum doses of MPH-IR and Ritalin-LA
           per day are 60 mg and of Concerta is 54 mg, as these are the maximum daily doses
           approved in Taiwan. The dose will be decreased should any clinically intolerable adverse
           conditions arise. The final titration dose of MPH with an acceptable response will be
           maintained through the follow-up period.

        3. Concomitant medications are prohibited. Compliance with the drug regimen will be
           verified at each visit based on reports by the patients' parents and how much of the
           prescription remains. Patients that start a new medication between the different
           time-points of the study will be considered dropouts.

        4. The ADHD Patients that decide not to receive medical treatment are usually reassessed by
           a child psychiatrist in the outpatient department once a month and may receive
           behavioral therapy or re-educative psychotherapy for the duration of the study.

        5. The healthy control subjects will be assessed only once, at the baseline. 5. Patients
           with ADHD will be reassessed at the twelfth month after baseline. The assessment
           procedures, including miRNAs analysis and ADHD measures, that were administered at
           baseline will be replicated.

      Year 2: Developing a SVM classification model and enrolling an independent validation group
      with 50 ADHD patients and 50 healthy control subjects (Testing Set)

        1. Based on the samples already gathered, the 10 most differentially expressed miRNAs
           between the ADHD group and control group can be determined. Those 10 values can be used
           to derive the SVM classification model and create a dependable and strong miRNA
           diagnosis panel for ADHD. Receiver operating characteristic (ROC) curves will be applied
           to evaluate the validity of this miRNA diagnosis panel. Both the specificity and
           sensitivity of this panel are expected to reach 80%.

        2. For the independent validation group (Testing Set), 50 eligible patients with ADHD and
           50 age- and gender-matched healthy control subjects will be recruited. Research
           psychiatrists will interview the participants using the K-SADS-E to confirm the
           diagnosis, and the participants will also undergo WISC-IV assessment to exclude
           intellectual disabilities.

        3. All ADHD patients and healthy control subjects will have experienced child psychologists
           administer Conners' CPT and Conners' CATA testing. The parents or guardians of the
           participants are required to complete the SNAP-IV parent form and the SAICA; the
           patient' teachers are required to complete the SNAP-IV teacher form; and the ADHD-RS and
           the CGI will be completed and rated, respectively, by a child psychiatrist.

        4. Venous blood will be collected from each participant using an EDTA anticoagulant tube.
           The blood samples will be further processed with the mirVana miRNA isolation kit (Life
           technology) to extract total RNAs. The levels of the 10 miRNAs elected for the SVM
           classification model will be determined by qRT-PCR.

        5. To verify the results of the Training Set, the miRNAs data of the Testing Set (both the
           50 ADHD patients and the 50 healthy control subjects) will be used for blind testing by
           the miRNA diagnosis panel. The discrimination validity expects both the sensitivity and
           specificity to reach 80%.

        6. The treatment and follow-up procedure for ADHD patients will be the same as in the first
           year. The 100 ADHD patients recruited in the first year will be followed and reevaluated

      Year 3: Completion of the 12-month follow-up of ADHD patients and performing analysis of
      target genes and enrichment pathways of the miRNAs

        1. No new participants will be recruited in the third year. The 12-month follow-up of all
           150 patients with ADHD will be completed during this year.

        2. The second assessment of patients with ADHD will be carried out to conclude this study
           (twelfth month after baseline). The same assessment procedures that were administered at
           baseline will be replicated.

      (1) Blood samples will be collected and be processed again to extract miRNAs-. (2) A research
      psychiatrist will interview the ADHD patients using the K-SADS-E in order to confirm that the
      patients still meet the diagnostic criteria of ADHD.

      (3) Patients with ADHD will undergo neuropsychological testing (WISC-IV, Conners CPT and
      Conners CATA) and have their behavioral symptoms rated (SNAP-IV, SAICA, ADHD-RS and CGI) by
      the same rater at baseline. To avoid any bias derived from medication, ADHD patients will be
      told not to take their medication on the day of the neuropsychological assessment.

      3. The trends of the miRNA levels during the 12-month follow-up will be identified. The
      differential miRNA levels between patients with versus without MPH treatment and between
      patients who continue versus those who remit from the ADHD diagnosis will be evaluated.
      Whether miRNAs can function as a biomarker that portrays the ADHD condition after treatment
      will be determined.

      4. The predicted target genes and their enrichment pathways of the differentially expressed
      miRNAs will be obtained using TargetScan v6.2, miRWalk, and DIANA-microT. The search will
      focus exclusively on the 3'-UTR regions of the target miRNAs with a p-value of 0.05 defining
      the probability distribution of random matches set in the software with a minimum miRNA seed
      length of 7.
    

Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        1. Patients with ADHD will be recruited from the out-patient department in Kaohsiung
             Chang Gung Memorial Hospital, Kaohsiung city, Taiwan

          2. Healthy controls will be recruited from the staff in Kaohsiung Chang Gung Memorial
             Hospital and from the communities in Kaohsiung city, Taiwan
      
Biospecimen description: 
      We hypothesize that miRNAs' expressions correlate significantly with the phenotypes and
      neurocognitive endophenotypes of ADHD
    
Keywords: attention-deficit/hyperactivity disorder; epigenetics; behavioral science; cognitive function; child and adolescent psychiatry