Exosomal MicroRNA Expression in Children With Autism Spectrum Disorder

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.