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2 patients with Prader-Willi Syndrome, exhibiting characteristic facial appearance, with an elongated face, thin upper lip and a prominent nose. a) a 15-year-old male. b) a 41-year-old female. This painting inspired the initial description of Angelman Syndrome

How a single genetic mutation can cause autism

New research shows the precise cell mechanisms that leads to autism disorder, and an existing drug which might help thousands of people with autism.

Last December it was announced that more than 1,000 gene mutations in people with autism had been identified, but it was left unexplained how these particular mutations increased the risk for autism. Now, researchers at the University of North Carolina (UNC) School of Medicine have found how one disabled molecular switch in one gene can lead to the disorder, while duplication or triplication of the same switch can lead to Angelman Syndrome (AS).

AS is a classic example of genomic imprinting. It is caused by deletion or inactivation of genes on the mother's chromosome 15. The father's copy of chromosome 15, which may be normal, is imprinted and therefore silenced. The companion syndrome, Prader-Willi Syndrome, is caused by a similar loss of a father's inherited genes and a mother's genes being imprinted and also silenced.

Ubiquitin-protein ligase E3A, UBE3A, is an enzyme produced by the UBE3A gene, to tag proteins for removal. This is a normal process that maintains healthy cell function by removing damaged or unnecessary proteins. The ligase attaches a small marker, ubiquitin, to unneeded proteins. At that point, structures called proteasomes, recognize and digest any protein so tagged.

UBE3A is switched off when it is tagged with a phosphate molecule. Deleting the function of UBE3A therefore causes cellular chaos. One type of neurodevelopmental disorder that results from this malfunction is Angelman syndrome (AS).  However, duplicating or triplicating UBE3A keeps UBE3A perpetually switched on. As a result, UBE3A becomes hyperactive, creating abnormal dendritic spines all over neurons and results in autism.

The research is published in the journal Cell

"Genetic studies are showing that there will be about 1,000 genes linked to autism. This means you could mutate any one of them and get the disorder. We found how [just] one of these mutations works."

Mark Zylka PhD, Associate Professor, Cell Biology and Physiology, UNC Neuroscience Center, senior author of the paper.

Research was conducted on human cell lines and in mouse models, as Zylka had cells from previous research with an autism-linked UBE3A mutation patient. Sequencing genes from those cell samples - including cells from the child's parents - Zulka found that only the child had hyperactive UBE3A, not the parents. The child's regulatory switch was broken causing UBE3A to be perpetually switched on and ubiquitin not being attached to proteins needing to be destroyed.

"When this child's mutation was introduced into an animal model, we saw all these dendritic spines form on the [mouse's] neurons," explained Zylka, a member of the Carolina Institute for Developmental Disabilities. "Having too many dendritic spines has been linked to autism." Their findings thus pointed to hyperactivation of UBE3A as the likely cause of the child's condition.

Duplication of the 15q chromosome region - which includes UBE3A and several other genes - is one of the most commonly seen genetic alterations in people with autism and is called "Dup15q Syndrome."

As part of their study, Zylka and team found Protein Kinase A (PKA) is the enzyme that tacks the phosphate group onto UBE3A. This finding has therapeutic implications, particularly as drugs exist to control PKA.

Zylka: "We think it may be possible to tamp down UBE3A in Dup15q patients to restore normal levels of enzyme activity in the brain. In fact, we tested known compounds and showed that two of them substantially reduced UBE3A activity in neurons."

One of the drugs, rolipram, previously had been tested in clinical trials treating depression but was discontinued when a patient suffered sudden death by epileptic seizure. In light of this result, Zylka believes retesting must be done with lower doses of rolipram, or any other drug that may increase PKA activity, if only to provide symptom relief for some Dup15q individuals. "The benefits might outweigh the risks," he feels. Such tests would be done with an animal model of Dup15q first.

While the bulk of this project focused on autism, it began by identifying Angelman Syndrome-linked mutations clustered in the same chromosome region where a phosphate group attaches to UBE3A. Zylka's team found that a number of Angelman mutations disrupt the function of UBE3A — and that deleting UBE3A function altogether causes cell chaos. Mutations that could essentially eliminate the UBE3A enzyme in Angelman Syndrome patients, is a discovery which could also help diagnose people with this rare and often misdiagnosed disorder.

Abstract Highlights
•PKA phosphorylates UBE3A at T485 and inhibits UBE3A ubiquitin ligase activity
•Autism-linked UBE3A T485A missense mutation disrupts phosphorylation regulation
•The T485A mutation hyperactivates UBE3A and increases synapse formation in vivo

Summary
Deletion of UBE3A causes the neurodevelopmental disorder Angelman syndrome (AS), while duplication or triplication of UBE3A is linked to autism. These genetic findings suggest that the ubiquitin ligase activity of UBE3A must be tightly maintained to promote normal brain development. Here, we found that protein kinase A (PKA) phosphorylates UBE3A in a region outside of the catalytic domain at residue T485 and inhibits UBE3A activity toward itself and other substrates. A de novo autism-linked missense mutation disrupts this phosphorylation site, causing enhanced UBE3A activity in vitro, enhanced substrate turnover in patient-derived cells, and excessive dendritic spine development in the brain. Our study identifies PKA as an upstream regulator of UBE3A activity and shows that an autism-linked mutation disrupts this phosphorylation control. Moreover, our findings implicate excessive UBE3A activity and the resulting synaptic dysfunction to autism pathogenesis.

Other co-authors included Ben Philpot, PhD, professor of cell biology and physiology, William Snider, MD, director of the UNC Neuroscience Center, and Klaus Hahn, PhD, the Ronald Thurman Distinguished Professor of Pharmacology. Janet Berrios, a graduate student at UNC, and Jason Newbern, PhD, assistant professor in the School of Life Sciences at Arizona State University, are also co-authors.

The National Institutes of Health, the Angelman Syndrome Foundation, The Foundation for Angelman Syndrome Therapeutics, and Autism Speaks funded this work.

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