Exosomal MicroRNA Expression in Children With Autism Spectrum Disorder
Condition: Autism Spectrum Disorder
MeSH terms: Autistic Disorder; Autism Spectrum Disorder; Child Development Disorders, Pervasive
Study type: Observational

Summary: 
      There is accumulating evidence that genetic expression plays a role in autism spectrum
      disorder, but the regulation of such genes is poorly understood. Small RNA particles, called
      microRNA (miRNA), have the ability to alter gene expression. These particles can be packaged
      and released from brain cells into the blood. Changes in miRNA may contribute to the patterns
      observed in autism spectrum disorder.

      The purpose of this study is to identify small RNA particles that regulate gene expression in
      autism spectrum disorder. The goal is to identify miRNA expression patterns which may improve
      our understanding and diagnosis of autism spectrum disorder.
    

Detailed description: 
      This study will recruit 20 children with idiopathic ASD (defined by DSM-IV criteria) and 20
      age- and gender-matched controls with typical neuropsychological developmental patterns. The
      number of subjects necessary for this study was determined by power analysis. Using a two
      sample t-test model with 15 ASD and 15 control subjects in each group would have 80% power to
      detect changes in expression equal to twice the standard deviation of the group mean with
      Bonferonni corrected level (p<0.0001) based on expectations of measuring 500 miRNA in each
      sample (Lenth, 2006).

      Parental consent and patient assent forms will be distributed to potential subjects at the
      University Pediatric and Adolescent Center and the Center for Development Behavior and
      Genetics. To explain the study, children will be told, "The doctors are doing a study about a
      disease called autism. If you want to be in this study a small amount of blood will be taken
      from your arm with a needle. The needle will hurt but will go away after a little while. The
      doctors will ask you to answer some questions about how you feel. You do not have to be in
      the study. No one will be mad at you if you don't want to do this."

      ASD subjects will be assessed using current methods in autism diagnosis: the Autism
      Diagnostic Observation Schedule (ADOS) and the Checklist for Autism in Toddlers (CHAT). The
      Vineland Behavior Scales will also be administered. All medical information collected will be
      coded and de-identified according to standard protocols.

      Venipuncture will be employed to collect 2ml of blood from each subject into an EDTA-free
      vacutainer tube. Samples will be left for 1 hour at 37˚C to allow clotting before storage at
      4˚C overnight. The sample will then be centrifuged at 4000 rpm for 20 minutes at 4˚C. Serum
      will be removed from the clot by pipetting and stored at -80˚C until miRNA extraction is
      performed.

      The decision to measure exosomal miRNA from serum is supported by a study of miRNA in 12
      human body fluids. Weber and colleagues (2010) determined that the median total concentration
      of RNA in plasma (308 ug/L) was greater than that of cerebrospinal fluid (111 ug/L).
      Furthermore, the authors found the number of detectable miRNAs in plasma (349) to be greater
      than cerebrospinal fluid (212). The miRNA content of plasma and cerebrospinal fluid has some
      intriguing similarities. Of the twenty most abundant miRNAs found in cerebrospinal fluid and
      plasma, 9 overlap. We have previously examined the expression of exosomal miRNA in the serum
      of human alcoholic subjects. Intriguingly, we characterized the expression levels of
      brain-specific miRNAs in serum samples from over 30% of those tested. This finding
      demonstrates that exosomal miRNAs from the central nervous system can be detected and
      quantified in human blood samples.

      Serum miRNA will be extracted using a Qiagen RNeasy Mini Kit according to manufacturer
      protocol. RNA will be processed with the Genisphere FlashTag HSR RNA labeling kit. RNA Spike
      Control Oligos, and poly(A) tails will be added to each RNA sample and a biotinylated
      signaling molecule will be ligated to the RNA. The labeled RNA samples will be added to a
      hybridization cocktail, incubated, and injected into Affymetrix miRNA 2.0 arrays. After 16
      hours of hybridization, the arrays will be washed and stained on an Affymetrix fluidics
      station 450 using protocol FS450_0003. Arrays will be scanned on an Affymetrix GeneChip
      Scanner 7G Plus. The cel files will be analyzed using miRNA QC Tool version 1.1.1.0.

      A 3-way ANOVA assessing the impact of ASD, age, and gender on miRNA expression will be
      employed along with a Mann Whitney Test. Targets with a P-value < 0.05 will be selected for
      further analysis after multiple testing correction with Bonferroni analysis. Significantly
      altered miRNAs will be queried on Mirbase.org for known mRNA targets. The target list will
      then be examined using DAVID bioinformatics functional annotation tool for enriched gene
      ontology categories. Correlations between miRNA expression and medical/neuropsychological
      characteristics will be calculated using Pearson Correlation statistics, filtering for miRNAs
      with p-value<0.05 and r>0.5.
    

Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Children from the University Pediatric and Adolescent Center and the Center for Development
        Behavior and Genetics in Syracuse, New York.
      
Biospecimen description: 
      Whole Blood
    
Keywords: Autism Spectrum Disorder; Exosomal miRNA expression