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Exome Study of Girls with Severe Autism Links New Genetic Variants to Disorder

NEW YORK (GenomeWeb) – By focusing on families with more than one girl with severe autism spectrum disorder, a Johns Hopkins University School of Medicine-led team homed in on four candidate genes linked to the disorder.

The Hopkins team and their colleagues sequenced the exomes of more than a dozen unrelated girls with severe autism to uncover genes harboring an excess of deleterious variants. One of these genes — CTNND2 — has been linked through functional analyses to both neuronal development and chromatin biology, as the researchers reported in Nature today.

"We've shown that even for genetically complicated diseases, families that have an extreme presentation are very informative in identifying culprit genes and their functions ­— or, as geneticists are taught, 'treasure your exceptions,'" senior author Aravinda Chakravarti from Hopkins said in a statement.

Though autism is a common neurodevelopmental disorder, it is more common among boys than girls, suggesting that girls with autism likely harbor more severe alleles that affect important developmental stages.

By focusing on families with more than one affected girl, Chakravarti and his colleagues reasoned that they would be better able to identify genes in which mutations impart a larger risk of developing autism.

"I think whom you sequence is as important — if not more so — than how many people are sequenced," he added.

He and his colleagues sequenced the exomes of 13 unrelated girls with severe autism and compared the resulting data to sequence data from 71 European females from the 1000 Genomes Project. They focused on missense variants absent from public databases as well as nonsense and splice site variants, leading to more than 3,000 variants of interest.

Four genes — CYFIP1, DLG1, PLXNA3, and CTNND2 — had an excess of deleterious variants and represented candidate genes for involvement in autism, the researchers reported.

A 2009 Nature paper on a genome-wide association study conducted by Chakravarti and his colleagues had implicated chromosome 5p in the disease, and as CTNND2 is located in that region, the researchers focused on that gene for their follow-up analyses in this study.

CTNND2, they noted, had two deleterious variants — G34S and R713C — both of which were not found in nearly 4,000 European controls from the 1000 Genomes project. The G34S variant was present at a low frequency among African ancestry samples in the Exome Variant Server and in one Luhyan sample, though none of the autism cases in the study were of African descent.

Next-generation sequencing of 362 additional girls with autism and analysis of the Simons Simplex Collection and 1000 Genomes Project uncovered an additional 11 CTNND2 variants, and the researchers noted that variants at conserved residues were more frequent in autism cases than in controls.

Chakravarti and his colleagues also uncovered an excess of exon-disrupting deletions affecting CTNND2 that they said implicates CTNND2 haploinsufficiency in autism.

Through functional assays using zebrafish and rodent models, the researchers found that CTNND2, when disrupted by G34S and R713C variants, disrupts the proper development of mammalian neural circuitry.

For instance, they found that primary hippocampal neurons from rat embryos expressing wild-type CTNND2 had higher dendritic spine density — dendritic spines are the primary sites of excitatory synapse formation — than ones with either G34S or R713C CTNND2.

In addition, some 71 other genes that are co-expressed with CTNND2 have also been implicated in autism, the researchers said. A pathway analysis of these genes indicated that they were enriched for involvement in dendrite morphogenesis and chromatin modification.

This, Chakravarti and his colleagues reported, indicates that CTNND2 is needed for the maintenance of spine structures and that the loss of its activity in null mice leads to fewer spines and a reduction in the levels of synaptic proteins, and likely to a reduction in the number of functional excitatory synapses.

"This is consistent with recent findings that many gene mutations associated with autism are involved in synapse development," co-author Richard Huganir from Hopkins added. "The results of this study add to the evidence that abnormal synaptic function may underlie the cognitive defects in autism."