Beyond genetics: What the new fields of functional genomics and epigenetics are revealing about autism
Today’s guest post comes to us from Valerie W. Hu, Ph.D. Dr. Valerie Hu is a Professor of Biochemistry and Molecular Biology at The George Washington University Medical Center as well as a mother of a son with ASD. She redirected her research focus towards autism about 5 years ago and has since published 6 papers on the genes and biological pathways associated with ASD. Dr. Hu received her Ph.D. in Chemistry from Caltech and did her postdoctoral research in Membrane Biochemistry and Immunology at UCLA. More information about her research and papers can be obtained at: http://www.gwumc.edu/biochem/faculty_vhu.html
For many years, genetics has followed the traditional approach to identifying genes associated with various disorders, including autism. However, the wide diversity of symptoms and behaviors associated with autism spectrum disorders (ASD) has posed a significant challenge to identifying mutations in one or a few genes that are reliably associated with ASD. More recent attention has focused on copy number variants (CNVs) which are submicroscopic regions of chromosomes that have been found to be lost or duplicated in some individuals with ASD. Together, these studies are making progress in identifying a number of genes that function at the synapse between nerve cells. Nevertheless, the combination of all known genetic mutations still does not account for the majority of autism cases or for the association of non-neurological symptoms observed in autistic individuals.
My laboratory at The George Washington University Medical Center has taken a functional genomics approach to studying genes that may be deregulated in autism. Rather than identify mutations in DNA, our goal has been to identify genes whose activity, as indicated by gene expression level, is altered in order to identify dysfunctional biochemical pathways and impaired cellular functions in autism. Thus, using a method known as DNA microarray analysis to profile gene expression in cells from identical twin pairs who are differentially diagnosed with autism (one autistic, the other not reaching the threshold for an autism diagnosis), sibling pairs where only one sibling is autistic, and unrelated case-controls, we identified many genes whose expression levels (activities) are different from non-autistic individuals. Furthermore, by subtyping the unrelated autistic individuals according to symptomatic severity across over 63 symptoms probed by a commonly used diagnostic assessment instrument (Autism Diagnostic Interview-Revised), we were able to identify gene “signatures” for each of 3 ASD subtypes studied. These signatures not only revealed genes unique to a given subtype of autism, but also overlapping genes that presumably control common symptoms of autism across subtypes. Interestingly, a set of genes that regulate the biological clock (that is, circadian rhythm) were found to be disrupted only in the subtype of ASD exhibiting severe language impairment.
These studies demonstrating different gene expression signatures between identical but differentially diagnosed twins as well as revealing differential expression of hundreds to thousands genes depending on ASD subtype suggest the involvement of “master switches” (or epigenetic mechanisms) in the control of gene expression. Two new studies by our laboratory, published in two different journals on Apr. 7, 2010, suggest that epigenetics may play a significant role in the regulation of gene expression in autism (read a blog from AS staff on epigenetics). In one study, published in the FASEB Journal, we identified chemical tags (called “methyl groups”) on the DNA of individuals with autism that led to gene silencing. This mode of turning off a gene is potentially reversible with the proper drugs if the specific gene can be properly targeted. The second study, which was published in the journal Genome Medicine, reports on the differential expression of microRNA in autism. MicroRNA are recently discovered snippets of RNA (ribonucleic acid), each of which can inhibit the expression (and thus activity) of hundreds of genes. The effects of microRNA are also reversible by treatment with complementary “anti-sense” RNA. While methylation inhibits gene expression at the level of DNA, microRNA inhibits at the level of RNA. These two studies together illustrate two different “epigenetic” mechanisms controlling gene activity in autism that lie beyond genetic mutations.
In the study published in the FASEB Journal, we again used cell lines derived from identical twins and sibling pairs in which only one of the twins or siblings was diagnosed with autism to identify chemical changes on DNA. We then compared the genes that showed changes in DNA tagging (methylation) with a list of genes that showed different levels of expression from these same individuals. The amounts of protein produced by two of the genes that appear on both lists were then investigated in brain tissues from the cerebellum and frontal cortex of autistic and control subjects which were obtained through the Autism Tissue Program. We found that both selected proteins, RORA (retinoic acid-related orphan receptor-alpha) and BCL-2, as predicted by the observed increase in methylation, were reduced in the autistic brain. Although BCL-2 has previously been reported to be reduced in autistic brain, RORA is a novel gene which is relevant to many of the observed deficits in autism. Specifically, RORA is involved in several key processes negatively affect by autism, including Purkinje cell differentiation, cerebellar development, protection of neurons against oxidative stress, suppression of inflammation, and regulation of circadian rhythm.
These results suggest that blocking the chemical tagging of these genes may reverse some symptoms of autism if targeted removal of methyl groups from specific genes can be accomplished. Furthermore, this study, which links molecular alterations in blood-derived cells to brain pathobiology, demonstrates the feasibility of using more easily accessible cells from blood (or other non-brain tissues) for diagnostic screening.
This research is reported in the study, titled “Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain,” which was recently published in the Federation of American Societies for Experimental Biology (FASEB) Journal, and is available online at: http://www.fasebj.org.
In the study published in Genome Medicine, we identified changes in the profile of microRNAs between twins and sibling pairs, again discordant for diagnosis of autism. We discovered that, despite using cells derived originally from blood, brain-specific and brain-related microRNAs were found to be differentially expressed in the autistic samples, and that these microRNAs could potentially regulate genes that control many processes known to be disrupted in autism. For example, differentially expressed miRNAs were found to target genes highly involved in neurological functions and disorders in addition to genes involved in gastrointestinal diseases, circadian rhythm signaling, and steroid hormone metabolism. The study further shows that by treating the cells with antisense RNA antagonists (inhibitors) to specific microRNA or by employing mimics of a particular microRNA, one can reverse the pattern of expression of a given target gene regulated by that microRNA.
This study, titled “Investigation of post-transcriptional gene regulatory networks associated with autism spectrum disorders by microRNA expression profiling of lymphoblastoid cell lines” was highlighted as an “Editor’s pick” in the journal Genome Medicine. It is available online at: http://genomemedicine.com/content/2/4/23.
By integrating both DNA methylation and miRNA expression studies with gene expression data, Dr. Hu and colleagues are applying a systems biology approach to understanding this complex disorder.
Finding appropriate treatments for autism is a challenge for families and clinicians alike. While behavioral treatments are an effective mainstay of therapeutic approaches, many individuals with autism benefit from the addition of medicinal interventions, particularly for problem behaviors, severe self-injury, and disruptive repetitive behaviors. A major impediment to finding effective treatment regimens is the fact that individual responses to the same medicine can vary greatly due to genetic background. Finding the most effective dose with the fewest side effects means slowly trying various doses, and possibly having to switch medicines. This is not only a challenge for the physician, but is also a confounding factor in large-scale clinical trials that aim to determine the overall effectiveness of a medication.