Exosomal MicroRNA Expression in Children With Autism Spectrum Disorder
| Status: | Not yet recruiting |
|---|---|
| Conditions: | Neurology, Psychiatric, Autism |
| Therapuetic Areas: | Neurology, Psychiatry / Psychology |
| Healthy: | No |
| Age Range: | 4 - 17 |
| Updated: | 2/7/2015 |
| Start Date: | January 2013 |
| Contact: | Steven Hicks, MD, PhD |
| Email: | hickss@upstate.edu |
| Phone: | 845 518 0649 |
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.
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.
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.
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.
Inclusion Criteria:
- Children ages 4-17
Exclusion Criteria:
- neurological impairments (i.e. cerebral palsy, epilepsy)
- sensory deficits (i.e. sensory or visual impairments)
- psychological disorders (i.e. obsessive compulsive disorder, attention deficit
disorder)
- control subjects with family history of autism spectrum disorder
- mental retardation
- history of preterm birth
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