<clinical_study>
  <!-- This xml conforms to an XML Schema at:
    https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
  <required_header>
    <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
    <link_text>Link to the current ClinicalTrials.gov record.</link_text>
    <url>https://clinicaltrials.gov/ct2/show/NCT03026309</url>
  </required_header>
  <id_info>
    <org_study_id>0112-16-ASMC</org_study_id>
    <nct_id>NCT03026309</nct_id>
  </id_info>
  <brief_title>PET-MRI F-DOPA Activity in the Mesocorticolimbic System and Depressive Symptoms in the Prediction of Treatment Compatibility</brief_title>
  <official_title>PET-MRI F-DOPA Activity in the Mesocorticolimbic System and Depressive Symptoms in the Prediction of Treatment Compatibility</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>Assuta Medical Center</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
  </sponsors>
  <source>Assuta Medical Center</source>
  <oversight_info>
    <has_dmc>No</has_dmc>
  </oversight_info>
  <brief_summary>
    <textblock>
      Main objectives: Comparing levels of F-DOPA reuptake rate as an indicator for Dopamine&#xD;
      metabolism of un-medicated Depressed patients to healthy individuals in the Mesocorticolimbic&#xD;
      System (VTA-NAc-PFC) and assessing structural differences between the two groups in the&#xD;
      Hippocampus, Hypothalamic-Pituitary gland and Mesocorticolimbic System (VTA-NAc-PFC) and&#xD;
      resting state fMRI.&#xD;
&#xD;
      Secondary objectives: 1. Comparing the differences of DNA Methylation in the plasma and serum&#xD;
      of patients compared to healthy controls. 2.Assessing the correlation between symptoms'&#xD;
      severity score (evaluated based on Hamilton Rating Scale) at base line and 6 months following&#xD;
      treatmnt to PET 18F-DOPA uptake repertoire in the Mesocorticolimbic System, structural&#xD;
      measurements and DNA Methylation.&#xD;
&#xD;
      Methodology: Study Design: A prospective, pilot study. 30 un medicated Depressed patients and&#xD;
      30 Healthy volunteers will perform a [18F] FDOPA PET/MRI scans following a HAM-D&#xD;
      questionnaire (Hamilton rating scale of depression) and blood tests.&#xD;
&#xD;
      PET-MR (Biograph mMR, Siemens AG, Erlangen, Germany) scans will be performed using the tracer&#xD;
      of dopamine precursor 3,4-dihydroxy-6-[18F]-fluoro-l-phenylalanine ([18F] FDOPA) that&#xD;
      reflects L-dopa transport, L-aromatic amino acid decarboxylase activity, vesicular uptake and&#xD;
      the number of dopamine nerve terminals. Measurements of dynamic F-DOPA parameters (Ki) and&#xD;
      quantitative measurements of static F-DOPA ( SUVmax and SUVmean) will be performed in the&#xD;
      ventral tagmental area (VTA), nucleus accombens (NAc) and pre-frontal cortex (PFC)which&#xD;
      comprise the Mesocorticolimbic System, bilaterally. MRI sequences of T1, T2 3D measurements&#xD;
      (assessing Volume) of the hippocampus, Hypothalamic-Pituitary gland and Mesocorticolimbic&#xD;
      system, DTI measurements in the mesocorticolimbic dopaminergic tract and BOLD (resting-state&#xD;
      f-MRI) in those brain networks. whole genome DNA Methylation from whole blood will be&#xD;
      performed.&#xD;
&#xD;
      To date there is no quantative standard of care evaluation tool that serves psychiatrists&#xD;
      when assigning medication for newly diagnosed depressed patients. The manner in which&#xD;
      medications are assigned are threw symptom evaluation and trial and error. Only a third of&#xD;
      the patients achieve remission after the first line of treatment. SSRI's are most common type&#xD;
      of medication used to treat Major Depression today. One third of patients remains un&#xD;
      responsive even after the fourth line of treatment (of different types of medications). Anti&#xD;
      depression medications way of action requires time to reach its effect and in many patients&#xD;
      with no avail or even causing symptom's severity. The PET-MR multimodality imaging tool&#xD;
      offers a cutting edge technology ideally fitted to measure brain disorders. The use of F-DOPA&#xD;
      radio-ligand with the PET-MR constitutes a novelty in the imaging of the depressed brain.&#xD;
      Dopamine is one of three of the monoamine neurotransmitters targeted by anti-depressive&#xD;
      medication, sharing metabolistic agents. dopamine has been proven to be connected to the&#xD;
      processing of emotion, motivation, hedonism and reward threw its action in the&#xD;
      Meso-cortico-limbic system. Epigenetics is a regulatory system that determines gene&#xD;
      expression. It is heritable in the one hand and reacts to environmental changes on the other.&#xD;
      It has been shown to be involved in psychiatric disorders.&#xD;
&#xD;
      PET-MR scans will be performed. DNA samples will be extracted from subject's whole blood&#xD;
      samples taken prior to scans. Then it will be analyzed for whole genome DNA methylation.&#xD;
      Taken together this novelty imaging technique and epigenetic mapping of peripheral markers&#xD;
      can be used to better understand and personalize anti-depressive treatment compatibility.&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      Introduction:&#xD;
&#xD;
      Major depressive disorder (MDD) is a versatile psychiatric disorder with a prevalence of 8-12&#xD;
      % of the population in many countries.&#xD;
&#xD;
      According to current approach MDD is characterized by lowered activity of monoamine&#xD;
      neurotransmitters, it is undetermined whether it is as a result of differences in sensitivity&#xD;
      of their receptors or depletion in the synaptic cleft due to low expression or mao-a/mao-b&#xD;
      hyperactivity.&#xD;
&#xD;
      Most antidepressant medication used today are based on the monoamine depletion theory of&#xD;
      depression. These medications include selective serotonine reuptake inhibitors (SSRIs),&#xD;
      serotonine norepinephrine reuptake inhibitors (SNRI's) or Tricyclics anti-depressants (TCAs),&#xD;
      all of which inhibit the reuptake of some or all of these monoamines, inducing elevated&#xD;
      levels in the synaptic cleft. Some medications affect the monoamines receptors directly.&#xD;
&#xD;
      Dysfunctional dopamine neurotransmission has an essential role in deficits with attention,&#xD;
      motivation and anhedonia which are core symptoms of depressive disorder. Pre-synaptic D2r&#xD;
      (Dopamine 2 receptor) density in the pre frontal cortex (PFC) has shown to be significantly&#xD;
      lower in depressed patients versus healthy individuals.&#xD;
&#xD;
      At present, most studies using imaging techniques in depressed patients use mainly different&#xD;
      types of magnetic resonance imaging (MRI) or separate nuclear medicine imaging with positron&#xD;
      emission tomography (PET) tracers. The role of traditional MRI in the evaluation of&#xD;
      depression and other psychiatric disorders, in different studies, is aimed mainly to&#xD;
      differentiate structural features and therefore help strengthening the diagnosis in that&#xD;
      aspect. There are several structural changes measured in the brains of MDD patients compared&#xD;
      to controls. Among them decreased hippocampal volume shown also compared to remitted&#xD;
      patients, increased Hypothalamic-Pituitary gland volume, decreased Pre Frontal Cortex (PFC)&#xD;
      and Basal-Ganglia volume. These findings are also established in accordance to the largest&#xD;
      meta-analysis study of structural changes using MRI and CT scans performed to date. PET scans&#xD;
      give indications regarding brain's general or agent specific metabolism. Most PET scans&#xD;
      performed on depressed patient uses FDG or 11-C Serotonine radioligands. Only a few studies&#xD;
      examine F-DOPA PET uptake in depressed patients, mostly with Parkinsonian depression and one&#xD;
      with affective flattening or impulsive symptoms on a very small sample size (n=6). Most of&#xD;
      the studies showing indication of decreased DOPA uptake in the striatum specifically in the&#xD;
      Nucleus Accombens (NAc). Additional information is required indicating F-DOPA uptake in&#xD;
      relevant regions of the brain in MDD. To the best of our knowledge this will be the first&#xD;
      study using [18F] F-DOPA PET/MRI in depressed patients. PET/MRI offers true multimodality&#xD;
      imaging by combining anatomy, function and molecular processes that will allow more accurate&#xD;
      identification of disease assessment.&#xD;
&#xD;
      A fact that is well established is that genetic factors together with environmental factors&#xD;
      occurring across the lifespan likely underlie the vulnerability for depression. However in a&#xD;
      mega-analysis of several genomewide association studies (GWAS), which analyzed 1.2 million&#xD;
      autosomal and X chromosome single-nucleotide polymorphisms (SNPs) found no SNP achieving&#xD;
      genome-wide significance. This discrepancy between the consistently observed substantial&#xD;
      heritability of major depression(18) and the fact no specific genetic variants have been&#xD;
      identified to robustly contribute to the disorder suggests other mechanisms at work.&#xD;
      Epigenetics refers to the changes in chromatin structure that underlies changes in gene&#xD;
      expression and that are not associated with alterations in DNA sequence. DNA methylation is&#xD;
      one of the systems underlies epigenetics. Methylation of the DNA requires the addition of a&#xD;
      methyl group at the 5' position of cytosines in CpG dinucleotides. Following cytosine&#xD;
      methylation, access of transcription factors to regulatory elements is reduced due to steric&#xD;
      interference. DNA methylation patterns are hereditable on the one hand and dynamically react&#xD;
      to the environment in the other hand. This trait of Epigenetics makes it a regulatory&#xD;
      mechanisms, determining gene expression, that is congruent to the nature of psychiatric&#xD;
      disorders. Diverse patterns of DNA methylation is observed in different tissues subjected to&#xD;
      different environments. It is even different between separate brain regions. An&#xD;
      Epigenome-wide association studies (EWAS) are on there way. In one of them in PFC tissue of&#xD;
      post mortem depressed subjects researchers found enrichment in neuronal growth and&#xD;
      developmental genes sites such as Brain drived neurotrophic factor .(BDNF) Decreased BDNF&#xD;
      levels are a well established marker for depression. Lower levels of BDNF protein have also&#xD;
      been reported in the hippocampus of animals exposed to chronic stress. Interestingly,&#xD;
      administration of antidepressants increased hippocampal BDNF, preventing the stress-induced&#xD;
      decrease. These findings are intriguing given the hippocampal volume loss observed in mood&#xD;
      disorders and decreased BDNF serum levels in individuals with mood disorders or depressive&#xD;
      personality traits. Based on these convergent preclinical and clinical data, the BDNF gene&#xD;
      represents a logical target for genetic investigations of mood disorders.&#xD;
&#xD;
      Another Epigenome-wide association study analyzing whole blood DNA found IL-6 and C-reactive&#xD;
      protein (CRP) plasma levels were increased in those persons with lifetime depression, and&#xD;
      among those with depression only, IL-6 methylation showed an inverse correlation with plasma&#xD;
      IL-6 and CRP.&#xD;
&#xD;
      To date there is no common imaging practice in the psychiatric clinic allowing&#xD;
      differentiating which medicated therapy is optimal for which patient. To the best of our&#xD;
      knowledge, this will be the first study to evaluate treatment compatibility using the&#xD;
      combination of [18F] F-DOPA PET/MRI imaging and symptoms assessment pre-treatment.&#xD;
&#xD;
      Study Objectives and Purpose:&#xD;
&#xD;
      The aim of the study is to assess the feasibility of the modality imaging and symptom&#xD;
      evaluation in the prediction of treatment compatibility. Dopamine metabolism measured in the&#xD;
      mesocorticolimbic system (VTA-NAc-PFC) by 18F-FDOPA PET with structural and functional MR&#xD;
      findings in patients diagnosed with depression pre-treatment.&#xD;
&#xD;
      Main objectives:&#xD;
&#xD;
      1. Comparing naïve levels of F-DOPA reuptake in healthy individuals to untreated Depressed&#xD;
      patients in the Mesocorticolimbic System (VTA-NAc-PFC) and assessing structural differences&#xD;
      between the two groups in the Hippocampus, Hypothalamic-Pituitary gland and Mesocorticolimbic&#xD;
      System (VTA-NAc-PFC) and resting state fMRI.&#xD;
&#xD;
      Secondary objectives:&#xD;
&#xD;
        1. Comparing the differences of DNA Methylation in the plasma of patients compared to&#xD;
           healthy controls.&#xD;
&#xD;
        2. Assessing the correlation between symptoms' severity score (evaluated based on Hamilton&#xD;
           Rating Scale) at base line and after 6 months to PET 18F-DOPA uptake repertoire in the&#xD;
           Mesocorticolimbic System, structural measurements and DNA Methylation.&#xD;
&#xD;
      Materials and Methods:&#xD;
&#xD;
      Study Design: A prospective, pilot study.&#xD;
&#xD;
      Clinical assessment:&#xD;
&#xD;
      A quantitative measure of symptoms' burden will be determined threw clinical assessment based&#xD;
      on &quot;Hamilton Rating Scale for Depression&quot; and based on DSM-V criteria at baseline&#xD;
      (pre-treatment), 3 months and 6 months after treatment initiation. According to DSM-5 the&#xD;
      diagnosis of depression requires five (or more) of the following symptoms that are present&#xD;
      during the same 2-week period and represent a change from previous functioning: (1) depressed&#xD;
      mood (2) loss of interest or pleasure. (3) Significant weight loss or weight. (4) Insomnia or&#xD;
      hypersomnia . (5) Psychomotor agitation or retardation.(6) Fatigue or loss of energy nearly.&#xD;
      (7) Feelings of worthlessness or excessive or inappropriate guilt.(8) Diminished ability to&#xD;
      think or concentrate. (9) Recurrent thoughts of death.&#xD;
&#xD;
      Symptom evaluation of Hamilton Rating Scale will be done by a psychiatrist. Finally we will&#xD;
      attempt to link specific clusters of symptoms to treatment compatibility in accordance to&#xD;
      imaging and molecular findings. The compatibility of treatment with these medication will be&#xD;
      compared to percentage of treatment success in the clinic in a 'standart of care' manner.&#xD;
      Assessment of the patient's progress will be determined by change in symptoms and/or a second&#xD;
      scan through follow up visits after 3,6 month.&#xD;
&#xD;
      .Imaging diagnostic PET/MR protocol: We would use the evaluation of the dopamine precursor&#xD;
      3,4-dihydroxy-6-[18F]-fluoro-l-phenylalanine ([18F] FDOPA) uptake rate constant (K(i)) that&#xD;
      reflects L-dopa transport. L-aromatic amino acid decarboxylase activity, vesicular uptake and&#xD;
      the number of dopamine nerve terminals. we would like to measure and compare the [18F] F-DOPA&#xD;
      uptake in the Mesocorticolimbic network in depressive unmedicated patients and healthy&#xD;
      individuals. We will then examine [18F] F-DOPA uptake repertoire and characterize a typical&#xD;
      spatiotemporal pattern along different depressive brains in accordance to the many kinds of&#xD;
      symptoms' manifestation and molecular and demographic data. In addition, we would implement,&#xD;
      new MR sequences such as diffuse tensor imaging (DTI) and susceptibility-weighted imaging&#xD;
      (SWI) to evaluate the mesocoticolimbic dopaminergic tract and use T1, T2 sequences to measure&#xD;
      Hippocampal and Hypothalamic-Pituitary gland volumes in these patients compared to healthy&#xD;
      individuals. Furthermore we will use T2* sequence to evaluate resting state&#xD;
      Blood-Oxygen-Level Dependent (BOLD) contrast imaging. In addition scan results will be&#xD;
      correlated to clinical evaluation of symptoms unmedicated patients.&#xD;
&#xD;
      All scans will be performed in the department of Nuclear Medicine at Tel-Aviv Assuta Medical&#xD;
      Center using a PET/MR scanner (Biograph mMR, Siemens AG, Erlangen, Germany) in accordance&#xD;
      with the manufacturer's guidelines.&#xD;
&#xD;
      Patients are required to fast at least 4 hours prior to arrival to the department. Upon&#xD;
      arrival an intravenous catheter will be placed for radiopharmaceutical and gadolinium&#xD;
      administration. Patients will receive an intravenous injection of 10 mCi of 18F-FDOPA on the&#xD;
      PET MR table and scanning will begin immediately. Dynamic PET parameters will be acquired&#xD;
      along with the different MR sequences. MR of the brain will include the following sequences:&#xD;
      T1, T2 and T2 fluid attenuated inversion recovery sequence (FLAIR), susceptibility-weighted&#xD;
      sequences (SWI), Diffuse Tensor Imaging (DTI), blood-oxygenation level-dependent (BOLD)&#xD;
      signals for resting-state functional imaging. In addition, perfusion of the brain will be&#xD;
      evaluated with and without gadolinium. Aa anatomical stereotactic map of the brain will be&#xD;
      layered in congruence to the scans using ExploreDTI Software.&#xD;
&#xD;
      For contrast-enhanced sequences we use Dotarem (gadoteric acid) (0.2 ml/kg ,0.1 mmol/kg at&#xD;
      2ml/s, 20ml saline flush) The total scan time will be about 45 minutes.&#xD;
&#xD;
      Data collection and procedures:&#xD;
&#xD;
      For eligible healthy volunteers, the following data will be recorded at baseline:&#xD;
&#xD;
        1. Baseline parameters: date of birth, sex, socioeconomic status, ethnicity, smoking,&#xD;
           alcohol consumption.&#xD;
&#xD;
        2. Detailed medical history.&#xD;
&#xD;
        3. List of medications&#xD;
&#xD;
        4. Symptom's Evaluation using Hamilton Rating Scale for Depression&#xD;
&#xD;
        5. Blood sample&#xD;
&#xD;
      For eligible depressed patients, the following data will be recorded at baseline, after 3&#xD;
      months and after 6 months.&#xD;
&#xD;
        1. Baseline parameters: date of birth, sex,&#xD;
&#xD;
        2. Detailed medical history.&#xD;
&#xD;
        3. List of medications&#xD;
&#xD;
        4. Symptom's Evaluation using Hamilton Rating Scale for Depression&#xD;
&#xD;
        5. Blood sample&#xD;
&#xD;
        6. Clinical diagnosis and symptoms onset.&#xD;
&#xD;
      Biological Sample collection:&#xD;
&#xD;
      Blood samples will be drawn from patients and healthy controls prior to scans for the&#xD;
      assessment of Whole genome DNA methylaion in plasma of peripheral blood. Blood samples will&#xD;
      be taken on from healthy controls and depreesed patients on base line. From depressed&#xD;
      subjects ,blood will be drawn olso after 6 months of treatment start date for the evaluation&#xD;
      of gene expression mRNA or protein of specific markers based on epigenetic findings from base&#xD;
      line.&#xD;
&#xD;
      Analysis of genome-wide promoter DNA methylation:&#xD;
&#xD;
      The procedure used for MeDIP analysis was adapted from previously published protocols.(13,14)&#xD;
      Brieﬂy, 2μg of DNA is sonicated, and methylated DNA is immunoprecipitated using&#xD;
      anti-5-methyl-cytosine (Eurogentec, Fremont, CA, USA). The DNA-antibody complex was&#xD;
      immunoprecipitated with protein G, and the methylated DNA is then resuspended in digestion&#xD;
      buffer (50mM TRisHCl pH8; 10mM EDTA; 0.5% SDS) and treated with proteinase K overnight at&#xD;
      55°C. The input and bound fraction is puriﬁed, ampliﬁed using the Whole Genome Ampliﬁcation&#xD;
      Kit (Sigma-Aldrich, St. Louis, MO, USA), and labeled for microarray hybridization with&#xD;
      Cy3-dUTP and Cy5-dUTP, respectively, using the CGH Enzymatic Labeling Kit (Agilent&#xD;
      Technologies, Mississauga, ON, Canada) in accordance with the manufacturer´s instructions.&#xD;
      Custom designed tiling arrays were used (Agilent Technologies). All steps of the&#xD;
      hybridization, washing, scanning and feature extraction procedures is performed in accordance&#xD;
      with the Agilent Technologies protocol for chipon-chip analysis. Extracted microarray&#xD;
      intensities is processed and analyzed using the R software environment for statistical&#xD;
      computing (http://www.r-project.org/).&#xD;
&#xD;
      Gene-speciﬁc Validation:&#xD;
&#xD;
      validation of MeDIP data will be performed applying quantitative-real time PCR (QPCR) using&#xD;
      the 2-ΔΔCt method. Data will be expressed as group means±s.e.m. The Graphpad 5 software will&#xD;
      be used to perform one-tailed Mann-Whitney U-tests.&#xD;
&#xD;
      RNA extraction from human blood:&#xD;
&#xD;
      blood for RNA extraction will be collected before injection of the radioligand and drawn into&#xD;
      PAXgene Blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland). RNA is extracted using the&#xD;
      PAXgene Blood RNA Kit (Qiagen). The quality and quantity of RNA samples will be analyzed&#xD;
      using an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany).&#xD;
&#xD;
      Analysis of gene expression in human blood:&#xD;
&#xD;
      Reverse transcription will be performed using the high-capacity cDNA Reverse Transcription&#xD;
      Kit (Life Technologies, Darmstadt, Germany). Gene expression levels will be analyzed using&#xD;
      QPCR and the 2-ΔΔCt method. Data will be expressed as group means±s.e.m. Statistical&#xD;
      signiﬁcance will be tested using one-tailed Mann-Whitney U-tests.&#xD;
&#xD;
      Pyrosequencing:&#xD;
&#xD;
      The promoter region of specific genes will be analyzed by pyrosequencing. In brief, fragments&#xD;
      of bisulﬁte-treated DNA(EpiTect Bisulﬁte Kit, Qiagen) will be ampliﬁed by PCR (HotStar Taq&#xD;
      DNA Polymerase, Qiagen) primer information using an unmodiﬁed forward primer and a&#xD;
      biotin-labeled reverse primer (Euroﬁns, Ebersberg, Germany). Pyrosequencing will be performed&#xD;
      using a PyroMark Q24 Advanced system (Qiagen; primer information see Supplementary Table S2)&#xD;
      in accordance with the manufacturer's protocol. Methylated and unmethylated EpiTect control&#xD;
      DNA samples (Qiagen) will be used as controls for bisulﬁte conversion, ampliﬁcation and&#xD;
      pyrosequencing. The percentage of methylation at each CpG site will be quantiﬁed using the&#xD;
      PyroMark Q24 Advanced software version 3.0.0 (Qiagen) Sequencing will be performed in&#xD;
      triplicate. Quality control ﬁltering and statistical analyses of the pyrosequencing results&#xD;
      will be conducted using R Version 2.15.3 (http://www.r-project.org). Measurements marked as&#xD;
      unreliable by the Pyromark software will be removed from the data set. Triplicate&#xD;
      measurements will be averaged after the removal of outliers (values deviating more than 3%).&#xD;
      A Mann-Whitney U-test will be used to compare the mean percentage of methylation of CpG sites&#xD;
      for the ELS versus control groups. Data will be presented as the mean±s. e.m.&#xD;
&#xD;
      Genetic analysis:&#xD;
&#xD;
      Genotypes will be investigated using GWAS data of a previous study of MDD. dbSNP of chosen&#xD;
      genes (http://www.ncbi.nlm.nih.gov/projects/SNP) will be searched for SNPs (single-nucleotide&#xD;
      polymorphisms) across the genes. The setbased test, as implemented in PLINK (v1.0.7)39 will&#xD;
      be performed (with default options and 105 permutations), to test for association between MDD&#xD;
      and the whole set of genetic variants in the data set&#xD;
&#xD;
      MR findings:&#xD;
&#xD;
        -  T1,T2 3D measurements (assessing Volume) of the hippocampus, Hypothalamic-Pituitary&#xD;
           gland and Mesocorticolimbic.&#xD;
&#xD;
        -  DTI measurements in the mesocorticolimbic dopaminergic tract.&#xD;
&#xD;
        -  Comparison of resting-state f-MRI (BOLD) brain networks in patients with depression with&#xD;
           normal maps&#xD;
&#xD;
      PET findings:&#xD;
&#xD;
        1. Visual assessment and quantitative measurements of static F-DOPA (e.g., SUVmax and&#xD;
           SUVmean) will be performed in the VTA, NAc, PFC bilaterally.&#xD;
&#xD;
        2. Measurements of dynamic F-DOPA parameters such as Ki when available (e.g., time to peak,&#xD;
           peak value etc.) will be measured.&#xD;
&#xD;
      Statistics:&#xD;
&#xD;
      Patient characteristics will be summarized using descriptive statistics. Quantitative&#xD;
      variables will be presented as mean and SD, qualitative variables will be presented as&#xD;
      frequencies.&#xD;
&#xD;
      Pearson correlation coefficient will be used to measure the strength of the relationship&#xD;
      between the PET in the different brain areas and the cluster symptoms.&#xD;
&#xD;
      A t-test will be used to compare the mean values of the different parameters. Sample size:&#xD;
      This is a pilot study for which a sample size of 60 subjects (30 healthy individuals and 30&#xD;
      patients) is required.&#xD;
&#xD;
      Participant Sampling: All patients fulfilling inclusion criteria and providing informed&#xD;
      consent will be consecutively enrolled.&#xD;
&#xD;
      Confidentiality:&#xD;
&#xD;
      All the data will be coded and subject will be identified only by the subject's initials and&#xD;
      by the subject's study number.&#xD;
&#xD;
      The coding will be done by the investigators and will be saved on their secured computers and&#xD;
      in the investigator file locked in a closet in the principal investigator office.&#xD;
&#xD;
      All reports and communications relating to subjects in the study will be using the code&#xD;
      given, with no personal information.&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Unknown status</overall_status>
  <last_known_status>Not yet recruiting</last_known_status>
  <start_date>March 2017</start_date>
  <completion_date type="Anticipated">March 2019</completion_date>
  <primary_completion_date type="Anticipated">March 2019</primary_completion_date>
  <study_type>Observational</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <observational_model>Cohort</observational_model>
    <time_perspective>Prospective</time_perspective>
  </study_design_info>
  <primary_outcome>
    <measure>3,4-dihydroxy-6-[18F]-fluoro-l-phenylalanine ([18F] FDOPA) uptake rate constant (K(i)) that reflects L-dopa transport.</measure>
    <time_frame>at base line</time_frame>
  </primary_outcome>
  <number_of_groups>2</number_of_groups>
  <enrollment type="Anticipated">60</enrollment>
  <condition>Depression</condition>
  <arm_group>
    <arm_group_label>depressed</arm_group_label>
    <description>un medicated diagnosed with major depression and scheduled to start SSRI treatment.</description>
  </arm_group>
  <arm_group>
    <arm_group_label>healthy</arm_group_label>
    <description>healthy volunteers.</description>
  </arm_group>
  <intervention>
    <intervention_type>Other</intervention_type>
    <intervention_name>no intervantion</intervention_name>
    <description>no intervention is being performed, only observational evaluations.</description>
    <arm_group_label>depressed</arm_group_label>
    <arm_group_label>healthy</arm_group_label>
  </intervention>
  <biospec_retention>Samples With DNA</biospec_retention>
  <biospec_descr>
    <textblock>
      whole blood.&#xD;
    </textblock>
  </biospec_descr>
  <eligibility>
    <study_pop>
      <textblock>
        Sample size: This is a pilot study for which a sample size of 60 subjects (30 healthy&#xD;
        individuals and 30 patients) is required.&#xD;
&#xD;
        Participant recruitment:&#xD;
&#xD;
        Eligible consecutive patients, satisfying the inclusion/exclusion criteria, will be&#xD;
        identified and recruited by the psychiatric clinic.&#xD;
&#xD;
        Eligible healthy volunteers will be recruited with assigned pamphlet approved by IRB.&#xD;
      </textblock>
    </study_pop>
    <sampling_method>Non-Probability Sample</sampling_method>
    <criteria>
      <textblock>
        -  Control group-30 healthy individuals:&#xD;
&#xD;
        Inclision criteria:&#xD;
&#xD;
          1. Adult male patients between the age of 30-50 years old.&#xD;
&#xD;
          2. Willing to participate and able to give there informed consent&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
          1. women.&#xD;
&#xD;
          2. With no history of psychiatric diagnosis.&#xD;
&#xD;
          3. History of any neurodegenerative disease or active oncologic disease.&#xD;
&#xD;
          4. With no history of psychiatric medication intake.&#xD;
&#xD;
          5. Patients treated with levodopa or any other medication known to interfere with the DAT&#xD;
             or catechol O-methyltransferase inhibitors, or with dopamine receptor blocking / or&#xD;
             catecholamine re-uptake blocking properties.&#xD;
&#xD;
          6. Contraindication to MR imaging.&#xD;
&#xD;
        study group- 30 Newly diagnosed depressed patient from the clinic of Dr. Lurie an extension&#xD;
        of Shalvata Mental health center .&#xD;
&#xD;
        Inclusion criteria:&#xD;
&#xD;
          1. Adult male patients between the age of 30-50 years old.&#xD;
&#xD;
          2. Patients willing to participate with all the study procedures and sign informed&#xD;
             consent form.&#xD;
&#xD;
          3. A clinical diagnosis of major depression.&#xD;
&#xD;
          4. With no history of psychiatric medication intake.&#xD;
&#xD;
          5. Scheduled to be treated with SSRI.&#xD;
&#xD;
        Exclusion criteria:&#xD;
&#xD;
          1. women.&#xD;
&#xD;
          2. Psychiatric patients diagnosed and treated with any psychiatric medication.&#xD;
&#xD;
          3. History of any neurodegenerative disease or active oncologic disease.&#xD;
&#xD;
          4. Patients with history of other brain disorder/pathology.&#xD;
&#xD;
          5. Patients treated with levodopa or any other medication known to interfere with the DAT&#xD;
             or catechol O-methyltransferase inhibitors, or with dopamine receptor blocking / or&#xD;
             catecholamine re-uptake blocking properties.&#xD;
&#xD;
          6. Contraindication to MR imaging.&#xD;
&#xD;
          7. With suicidal symptoms.&#xD;
&#xD;
        Subject withdrawal criteria:&#xD;
&#xD;
          1. Subject who withdrawn his consent at any point of the study.&#xD;
&#xD;
          2. Inability to perform MR&#xD;
&#xD;
          3. Non compliant with treatment intake or follow up observations.&#xD;
&#xD;
          4. Any decision made by the investigator that termination is in subject's best intrest.&#xD;
&#xD;
          5. Serius adverse event relating to the study.&#xD;
&#xD;
             -&#xD;
      </textblock>
    </criteria>
    <gender>Male</gender>
    <minimum_age>30 Years</minimum_age>
    <maximum_age>50 Years</maximum_age>
    <healthy_volunteers>Accepts Healthy Volunteers</healthy_volunteers>
  </eligibility>
  <overall_contact>
    <last_name>david groshar, MD</last_name>
    <phone>+972-37644364</phone>
    <email>davidg@assuta.co.il</email>
  </overall_contact>
  <overall_contact_backup>
    <last_name>Noa Zifman, MSc</last_name>
    <phone>+972-523755213</phone>
    <email>noaz@assuta.co.il</email>
  </overall_contact_backup>
  <verification_date>January 2017</verification_date>
  <study_first_submitted>January 18, 2017</study_first_submitted>
  <study_first_submitted_qc>January 18, 2017</study_first_submitted_qc>
  <study_first_posted type="Estimate">January 20, 2017</study_first_posted>
  <last_update_submitted>January 18, 2017</last_update_submitted>
  <last_update_submitted_qc>January 18, 2017</last_update_submitted_qc>
  <last_update_posted type="Estimate">January 20, 2017</last_update_posted>
  <responsible_party>
    <responsible_party_type>Principal Investigator</responsible_party_type>
    <investigator_affiliation>Assuta Medical Center</investigator_affiliation>
    <investigator_full_name>david groshar</investigator_full_name>
    <investigator_title>david groshar</investigator_title>
  </responsible_party>
  <condition_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Depression</mesh_term>
  </condition_browse>
  <patient_data>
    <sharing_ipd>No</sharing_ipd>
  </patient_data>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


download date: ClinicalTrials.gov processed this data on September 20, 2023
link text: Link to the current ClinicalTrials.gov record.
url: https://clinicaltrials.gov/ct2/show/NCT03026309
org study id: 0112-16-ASMC
nct id: NCT03026309
lead sponsor: 
has dmc: No
Main objectives: Comparing levels of F-DOPA reuptake rate as an indicator for Dopamine
      metabolism of un-medicated Depressed patients to healthy individuals in the Mesocorticolimbic
      System (VTA-NAc-PFC) and assessing structural differences between the two groups in the
      Hippocampus, Hypothalamic-Pituitary gland and Mesocorticolimbic System (VTA-NAc-PFC) and
      resting state fMRI.

      Secondary objectives: 1. Comparing the differences of DNA Methylation in the plasma and serum
      of patients compared to healthy controls. 2.Assessing the correlation between symptoms'
      severity score (evaluated based on Hamilton Rating Scale) at base line and 6 months following
      treatmnt to PET 18F-DOPA uptake repertoire in the Mesocorticolimbic System, structural
      measurements and DNA Methylation.

      Methodology: Study Design: A prospective, pilot study. 30 un medicated Depressed patients and
      30 Healthy volunteers will perform a [18F] FDOPA PET/MRI scans following a HAM-D
      questionnaire (Hamilton rating scale of depression) and blood tests.

      PET-MR (Biograph mMR, Siemens AG, Erlangen, Germany) scans will be performed using the tracer
      of dopamine precursor 3,4-dihydroxy-6-[18F]-fluoro-l-phenylalanine ([18F] FDOPA) that
      reflects L-dopa transport, L-aromatic amino acid decarboxylase activity, vesicular uptake and
      the number of dopamine nerve terminals. Measurements of dynamic F-DOPA parameters (Ki) and
      quantitative measurements of static F-DOPA ( SUVmax and SUVmean) will be performed in the
      ventral tagmental area (VTA), nucleus accombens (NAc) and pre-frontal cortex (PFC)which
      comprise the Mesocorticolimbic System, bilaterally. MRI sequences of T1, T2 3D measurements
      (assessing Volume) of the hippocampus, Hypothalamic-Pituitary gland and Mesocorticolimbic
      system, DTI measurements in the mesocorticolimbic dopaminergic tract and BOLD (resting-state
      f-MRI) in those brain networks. whole genome DNA Methylation from whole blood will be
      performed.

      To date there is no quantative standard of care evaluation tool that serves psychiatrists
      when assigning medication for newly diagnosed depressed patients. The manner in which
      medications are assigned are threw symptom evaluation and trial and error. Only a third of
      the patients achieve remission after the first line of treatment. SSRI's are most common type
      of medication used to treat Major Depression today. One third of patients remains un
      responsive even after the fourth line of treatment (of different types of medications). Anti
      depression medications way of action requires time to reach its effect and in many patients
      with no avail or even causing symptom's severity. The PET-MR multimodality imaging tool
      offers a cutting edge technology ideally fitted to measure brain disorders. The use of F-DOPA
      radio-ligand with the PET-MR constitutes a novelty in the imaging of the depressed brain.
      Dopamine is one of three of the monoamine neurotransmitters targeted by anti-depressive
      medication, sharing metabolistic agents. dopamine has been proven to be connected to the
      processing of emotion, motivation, hedonism and reward threw its action in the
      Meso-cortico-limbic system. Epigenetics is a regulatory system that determines gene
      expression. It is heritable in the one hand and reacts to environmental changes on the other.
      It has been shown to be involved in psychiatric disorders.

      PET-MR scans will be performed. DNA samples will be extracted from subject's whole blood
      samples taken prior to scans. Then it will be analyzed for whole genome DNA methylation.
      Taken together this novelty imaging technique and epigenetic mapping of peripheral markers
      can be used to better understand and personalize anti-depressive treatment compatibility.
Introduction:

      Major depressive disorder (MDD) is a versatile psychiatric disorder with a prevalence of 8-12
      % of the population in many countries.

      According to current approach MDD is characterized by lowered activity of monoamine
      neurotransmitters, it is undetermined whether it is as a result of differences in sensitivity
      of their receptors or depletion in the synaptic cleft due to low expression or mao-a/mao-b
      hyperactivity.

      Most antidepressant medication used today are based on the monoamine depletion theory of
      depression. These medications include selective serotonine reuptake inhibitors (SSRIs),
      serotonine norepinephrine reuptake inhibitors (SNRI's) or Tricyclics anti-depressants (TCAs),
      all of which inhibit the reuptake of some or all of these monoamines, inducing elevated
      levels in the synaptic cleft. Some medications affect the monoamines receptors directly.

      Dysfunctional dopamine neurotransmission has an essential role in deficits with attention,
      motivation and anhedonia which are core symptoms of depressive disorder. Pre-synaptic D2r
      (Dopamine 2 receptor) density in the pre frontal cortex (PFC) has shown to be significantly
      lower in depressed patients versus healthy individuals.

      At present, most studies using imaging techniques in depressed patients use mainly different
      types of magnetic resonance imaging (MRI) or separate nuclear medicine imaging with positron
      emission tomography (PET) tracers. The role of traditional MRI in the evaluation of
      depression and other psychiatric disorders, in different studies, is aimed mainly to
      differentiate structural features and therefore help strengthening the diagnosis in that
      aspect. There are several structural changes measured in the brains of MDD patients compared
      to controls. Among them decreased hippocampal volume shown also compared to remitted
      patients, increased Hypothalamic-Pituitary gland volume, decreased Pre Frontal Cortex (PFC)
      and Basal-Ganglia volume. These findings are also established in accordance to the largest
      meta-analysis study of structural changes using MRI and CT scans performed to date. PET scans
      give indications regarding brain's general or agent specific metabolism. Most PET scans
      performed on depressed patient uses FDG or 11-C Serotonine radioligands. Only a few studies
      examine F-DOPA PET uptake in depressed patients, mostly with Parkinsonian depression and one
      with affective flattening or impulsive symptoms on a very small sample size (n=6). Most of
      the studies showing indication of decreased DOPA uptake in the striatum specifically in the
      Nucleus Accombens (NAc). Additional information is required indicating F-DOPA uptake in
      relevant regions of the brain in MDD. To the best of our knowledge this will be the first
      study using [18F] F-DOPA PET/MRI in depressed patients. PET/MRI offers true multimodality
      imaging by combining anatomy, function and molecular processes that will allow more accurate
      identification of disease assessment.

      A fact that is well established is that genetic factors together with environmental factors
      occurring across the lifespan likely underlie the vulnerability for depression. However in a
      mega-analysis of several genomewide association studies (GWAS), which analyzed 1.2 million
      autosomal and X chromosome single-nucleotide polymorphisms (SNPs) found no SNP achieving
      genome-wide significance. This discrepancy between the consistently observed substantial
      heritability of major depression(18) and the fact no specific genetic variants have been
      identified to robustly contribute to the disorder suggests other mechanisms at work.
      Epigenetics refers to the changes in chromatin structure that underlies changes in gene
      expression and that are not associated with alterations in DNA sequence. DNA methylation is
      one of the systems underlies epigenetics. Methylation of the DNA requires the addition of a
      methyl group at the 5' position of cytosines in CpG dinucleotides. Following cytosine
      methylation, access of transcription factors to regulatory elements is reduced due to steric
      interference. DNA methylation patterns are hereditable on the one hand and dynamically react
      to the environment in the other hand. This trait of Epigenetics makes it a regulatory
      mechanisms, determining gene expression, that is congruent to the nature of psychiatric
      disorders. Diverse patterns of DNA methylation is observed in different tissues subjected to
      different environments. It is even different between separate brain regions. An
      Epigenome-wide association studies (EWAS) are on there way. In one of them in PFC tissue of
      post mortem depressed subjects researchers found enrichment in neuronal growth and
      developmental genes sites such as Brain drived neurotrophic factor .(BDNF) Decreased BDNF
      levels are a well established marker for depression. Lower levels of BDNF protein have also
      been reported in the hippocampus of animals exposed to chronic stress. Interestingly,
      administration of antidepressants increased hippocampal BDNF, preventing the stress-induced
      decrease. These findings are intriguing given the hippocampal volume loss observed in mood
      disorders and decreased BDNF serum levels in individuals with mood disorders or depressive
      personality traits. Based on these convergent preclinical and clinical data, the BDNF gene
      represents a logical target for genetic investigations of mood disorders.

      Another Epigenome-wide association study analyzing whole blood DNA found IL-6 and C-reactive
      protein (CRP) plasma levels were increased in those persons with lifetime depression, and
      among those with depression only, IL-6 methylation showed an inverse correlation with plasma
      IL-6 and CRP.

      To date there is no common imaging practice in the psychiatric clinic allowing
      differentiating which medicated therapy is optimal for which patient. To the best of our
      knowledge, this will be the first study to evaluate treatment compatibility using the
      combination of [18F] F-DOPA PET/MRI imaging and symptoms assessment pre-treatment.

      Study Objectives and Purpose:

      The aim of the study is to assess the feasibility of the modality imaging and symptom
      evaluation in the prediction of treatment compatibility. Dopamine metabolism measured in the
      mesocorticolimbic system (VTA-NAc-PFC) by 18F-FDOPA PET with structural and functional MR
      findings in patients diagnosed with depression pre-treatment.

      Main objectives:

      1. Comparing naïve levels of F-DOPA reuptake in healthy individuals to untreated Depressed
      patients in the Mesocorticolimbic System (VTA-NAc-PFC) and assessing structural differences
      between the two groups in the Hippocampus, Hypothalamic-Pituitary gland and Mesocorticolimbic
      System (VTA-NAc-PFC) and resting state fMRI.

      Secondary objectives:

        1. Comparing the differences of DNA Methylation in the plasma of patients compared to
           healthy controls.

        2. Assessing the correlation between symptoms' severity score (evaluated based on Hamilton
           Rating Scale) at base line and after 6 months to PET 18F-DOPA uptake repertoire in the
           Mesocorticolimbic System, structural measurements and DNA Methylation.

      Materials and Methods:

      Study Design: A prospective, pilot study.

      Clinical assessment:

      A quantitative measure of symptoms' burden will be determined threw clinical assessment based
      on "Hamilton Rating Scale for Depression" and based on DSM-V criteria at baseline
      (pre-treatment), 3 months and 6 months after treatment initiation. According to DSM-5 the
      diagnosis of depression requires five (or more) of the following symptoms that are present
      during the same 2-week period and represent a change from previous functioning: (1) depressed
      mood (2) loss of interest or pleasure. (3) Significant weight loss or weight. (4) Insomnia or
      hypersomnia . (5) Psychomotor agitation or retardation.(6) Fatigue or loss of energy nearly.
      (7) Feelings of worthlessness or excessive or inappropriate guilt.(8) Diminished ability to
      think or concentrate. (9) Recurrent thoughts of death.

      Symptom evaluation of Hamilton Rating Scale will be done by a psychiatrist. Finally we will
      attempt to link specific clusters of symptoms to treatment compatibility in accordance to
      imaging and molecular findings. The compatibility of treatment with these medication will be
      compared to percentage of treatment success in the clinic in a 'standart of care' manner.
      Assessment of the patient's progress will be determined by change in symptoms and/or a second
      scan through follow up visits after 3,6 month.

      .Imaging diagnostic PET/MR protocol: We would use the evaluation of the dopamine precursor
      3,4-dihydroxy-6-[18F]-fluoro-l-phenylalanine ([18F] FDOPA) uptake rate constant (K(i)) that
      reflects L-dopa transport. L-aromatic amino acid decarboxylase activity, vesicular uptake and
      the number of dopamine nerve terminals. we would like to measure and compare the [18F] F-DOPA
      uptake in the Mesocorticolimbic network in depressive unmedicated patients and healthy
      individuals. We will then examine [18F] F-DOPA uptake repertoire and characterize a typical
      spatiotemporal pattern along different depressive brains in accordance to the many kinds of
      symptoms' manifestation and molecular and demographic data. In addition, we would implement,
      new MR sequences such as diffuse tensor imaging (DTI) and susceptibility-weighted imaging
      (SWI) to evaluate the mesocoticolimbic dopaminergic tract and use T1, T2 sequences to measure
      Hippocampal and Hypothalamic-Pituitary gland volumes in these patients compared to healthy
      individuals. Furthermore we will use T2* sequence to evaluate resting state
      Blood-Oxygen-Level Dependent (BOLD) contrast imaging. In addition scan results will be
      correlated to clinical evaluation of symptoms unmedicated patients.

      All scans will be performed in the department of Nuclear Medicine at Tel-Aviv Assuta Medical
      Center using a PET/MR scanner (Biograph mMR, Siemens AG, Erlangen, Germany) in accordance
      with the manufacturer's guidelines.

      Patients are required to fast at least 4 hours prior to arrival to the department. Upon
      arrival an intravenous catheter will be placed for radiopharmaceutical and gadolinium
      administration. Patients will receive an intravenous injection of 10 mCi of 18F-FDOPA on the
      PET MR table and scanning will begin immediately. Dynamic PET parameters will be acquired
      along with the different MR sequences. MR of the brain will include the following sequences:
      T1, T2 and T2 fluid attenuated inversion recovery sequence (FLAIR), susceptibility-weighted
      sequences (SWI), Diffuse Tensor Imaging (DTI), blood-oxygenation level-dependent (BOLD)
      signals for resting-state functional imaging. In addition, perfusion of the brain will be
      evaluated with and without gadolinium. Aa anatomical stereotactic map of the brain will be
      layered in congruence to the scans using ExploreDTI Software.

      For contrast-enhanced sequences we use Dotarem (gadoteric acid) (0.2 ml/kg ,0.1 mmol/kg at
      2ml/s, 20ml saline flush) The total scan time will be about 45 minutes.

      Data collection and procedures:

      For eligible healthy volunteers, the following data will be recorded at baseline:

        1. Baseline parameters: date of birth, sex, socioeconomic status, ethnicity, smoking,
           alcohol consumption.

        2. Detailed medical history.

        3. List of medications

        4. Symptom's Evaluation using Hamilton Rating Scale for Depression

        5. Blood sample

      For eligible depressed patients, the following data will be recorded at baseline, after 3
      months and after 6 months.

        1. Baseline parameters: date of birth, sex,

        2. Detailed medical history.

        3. List of medications

        4. Symptom's Evaluation using Hamilton Rating Scale for Depression

        5. Blood sample

        6. Clinical diagnosis and symptoms onset.

      Biological Sample collection:

      Blood samples will be drawn from patients and healthy controls prior to scans for the
      assessment of Whole genome DNA methylaion in plasma of peripheral blood. Blood samples will
      be taken on from healthy controls and depreesed patients on base line. From depressed
      subjects ,blood will be drawn olso after 6 months of treatment start date for the evaluation
      of gene expression mRNA or protein of specific markers based on epigenetic findings from base
      line.

      Analysis of genome-wide promoter DNA methylation:

      The procedure used for MeDIP analysis was adapted from previously published protocols.(13,14)
      Brieﬂy, 2μg of DNA is sonicated, and methylated DNA is immunoprecipitated using
      anti-5-methyl-cytosine (Eurogentec, Fremont, CA, USA). The DNA-antibody complex was
      immunoprecipitated with protein G, and the methylated DNA is then resuspended in digestion
      buffer (50mM TRisHCl pH8; 10mM EDTA; 0.5% SDS) and treated with proteinase K overnight at
      55°C. The input and bound fraction is puriﬁed, ampliﬁed using the Whole Genome Ampliﬁcation
      Kit (Sigma-Aldrich, St. Louis, MO, USA), and labeled for microarray hybridization with
      Cy3-dUTP and Cy5-dUTP, respectively, using the CGH Enzymatic Labeling Kit (Agilent
      Technologies, Mississauga, ON, Canada) in accordance with the manufacturer´s instructions.
      Custom designed tiling arrays were used (Agilent Technologies). All steps of the
      hybridization, washing, scanning and feature extraction procedures is performed in accordance
      with the Agilent Technologies protocol for chipon-chip analysis. Extracted microarray
      intensities is processed and analyzed using the R software environment for statistical
      computing (http://www.r-project.org/).

      Gene-speciﬁc Validation:

      validation of MeDIP data will be performed applying quantitative-real time PCR (QPCR) using
      the 2-ΔΔCt method. Data will be expressed as group means±s.e.m. The Graphpad 5 software will
      be used to perform one-tailed Mann-Whitney U-tests.

      RNA extraction from human blood:

      blood for RNA extraction will be collected before injection of the radioligand and drawn into
      PAXgene Blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland). RNA is extracted using the
      PAXgene Blood RNA Kit (Qiagen). The quality and quantity of RNA samples will be analyzed
      using an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany).

      Analysis of gene expression in human blood:

      Reverse transcription will be performed using the high-capacity cDNA Reverse Transcription
      Kit (Life Technologies, Darmstadt, Germany). Gene expression levels will be analyzed using
      QPCR and the 2-ΔΔCt method. Data will be expressed as group means±s.e.m. Statistical
      signiﬁcance will be tested using one-tailed Mann-Whitney U-tests.

      Pyrosequencing:

      The promoter region of specific genes will be analyzed by pyrosequencing. In brief, fragments
      of bisulﬁte-treated DNA(EpiTect Bisulﬁte Kit, Qiagen) will be ampliﬁed by PCR (HotStar Taq
      DNA Polymerase, Qiagen) primer information using an unmodiﬁed forward primer and a
      biotin-labeled reverse primer (Euroﬁns, Ebersberg, Germany). Pyrosequencing will be performed
      using a PyroMark Q24 Advanced system (Qiagen; primer information see Supplementary Table S2)
      in accordance with the manufacturer's protocol. Methylated and unmethylated EpiTect control
      DNA samples (Qiagen) will be used as controls for bisulﬁte conversion, ampliﬁcation and
      pyrosequencing. The percentage of methylation at each CpG site will be quantiﬁed using the
      PyroMark Q24 Advanced software version 3.0.0 (Qiagen) Sequencing will be performed in
      triplicate. Quality control ﬁltering and statistical analyses of the pyrosequencing results
      will be conducted using R Version 2.15.3 (http://www.r-project.org). Measurements marked as
      unreliable by the Pyromark software will be removed from the data set. Triplicate
      measurements will be averaged after the removal of outliers (values deviating more than 3%).
      A Mann-Whitney U-test will be used to compare the mean percentage of methylation of CpG sites
      for the ELS versus control groups. Data will be presented as the mean±s. e.m.

      Genetic analysis:

      Genotypes will be investigated using GWAS data of a previous study of MDD. dbSNP of chosen
      genes (http://www.ncbi.nlm.nih.gov/projects/SNP) will be searched for SNPs (single-nucleotide
      polymorphisms) across the genes. The setbased test, as implemented in PLINK (v1.0.7)39 will
      be performed (with default options and 105 permutations), to test for association between MDD
      and the whole set of genetic variants in the data set

      MR findings:

        -  T1,T2 3D measurements (assessing Volume) of the hippocampus, Hypothalamic-Pituitary
           gland and Mesocorticolimbic.

        -  DTI measurements in the mesocorticolimbic dopaminergic tract.

        -  Comparison of resting-state f-MRI (BOLD) brain networks in patients with depression with
           normal maps

      PET findings:

        1. Visual assessment and quantitative measurements of static F-DOPA (e.g., SUVmax and
           SUVmean) will be performed in the VTA, NAc, PFC bilaterally.

        2. Measurements of dynamic F-DOPA parameters such as Ki when available (e.g., time to peak,
           peak value etc.) will be measured.

      Statistics:

      Patient characteristics will be summarized using descriptive statistics. Quantitative
      variables will be presented as mean and SD, qualitative variables will be presented as
      frequencies.

      Pearson correlation coefficient will be used to measure the strength of the relationship
      between the PET in the different brain areas and the cluster symptoms.

      A t-test will be used to compare the mean values of the different parameters. Sample size:
      This is a pilot study for which a sample size of 60 subjects (30 healthy individuals and 30
      patients) is required.

      Participant Sampling: All patients fulfilling inclusion criteria and providing informed
      consent will be consecutively enrolled.

      Confidentiality:

      All the data will be coded and subject will be identified only by the subject's initials and
      by the subject's study number.

      The coding will be done by the investigators and will be saved on their secured computers and
      in the investigator file locked in a closet in the principal investigator office.

      All reports and communications relating to subjects in the study will be using the code
      given, with no personal information.
observational model: Cohort
time perspective: Prospective
measure: 3,4-dihydroxy-6-[18F]-fluoro-l-phenylalanine ([18F] FDOPA) uptake rate constant (K(i)) that reflects L-dopa transport.
time frame: at base line
arm group label: depressed
description: un medicated diagnosed with major depression and scheduled to start SSRI treatment.
arm group label: healthy
description: healthy volunteers.
intervention type: Other
intervention name: no intervantion
description: no intervention is being performed, only observational evaluations.
arm group label: depressed
arm group label: healthy
whole blood.
study pop: 
sampling method: Non-Probability Sample
criteria: 
gender: Male
minimum age: 30 Years
maximum age: 50 Years
healthy volunteers: Accepts Healthy Volunteers
last name: david groshar, MD
phone: +972-37644364
email: davidg@assuta.co.il
last name: Noa Zifman, MSc
phone: +972-523755213
email: noaz@assuta.co.il
responsible party type: Principal Investigator
investigator affiliation: Assuta Medical Center
investigator full name: david groshar
investigator title: david groshar
mesh term: Depression
sharing ipd: No