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Autism-Associated Variants Uncovered by Population Diagnostics, SickKids Team Could Yield Dx Tests

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Researchers who set out to compare the ability of SNP and comparative genomic hybridization microarray platforms to detect copy number variation in autusm spectrum disorder managed to identify 16 genes that hadn't been associated with ASD before.

The results of the study, a joint project between the Hospital for Sick Children, or SickKids, in Toronto and Population Diagnostics, a privately held, Melville, NY-based company that is developing a test for early-stage ASD diagnosis, were published online in the journal G3 this week.

According to the authors, the new study has implications for array users, as the majority of CNVs identified with the CGH array used in the study — a million-feature chip manufactured by Agilent Technologies — had been missed in previous studies that relied on high-density SNP microarray platforms.

The variants identified in the study may also find their way into the clinic, Jim Chinitz, CEO of PDx, told BioArray News this week. Though Chinitz declined to reveal the terms of any contract with SickKids, Chinitz said that it is "implicit in any industry-academic relationship" that a firm like PDx would "endeavor to commercialize discoveries with protections in place" that would enable the firm to "address unmet medical needs and to do this ahead of potential competitors."

Founded in 2006, PDx's various commercial activities are driven by its method of comparing CNVs in subjects with a given condition to those in healthy individuals. By eliminating benign and irrelevant CNVs in such a comparison, researchers are able to identify genes that are pathogenic and can then be interrogated using higher resolution genetic analysis techniques such as sequencing, according to the firm.

The company is developing a number of tests based on its approach, including assays for neurodegenerative diseases, such as Parkinson's and Alzheimer's, food allergies, and drug discovery. In addition to these endeavors, the company since its founding has focused on identifying genetic variants that can be used to diagnose autism at an early stage.

As part of that effort, PDx has been working with Stephen Scherer, director of the Center for Applied Genomics at SickKids, to "discover as many new genes for autism as possible," Chinitz said. Scherer's lab has been prolific when it comes to autism research. So far this year, Scherer has co-authored a dozen papers related to ASD, most recently a Human Molecular Genetics paper that argued that individual common variants exert weak effects on the risk for autism.

"PDx and SickKids have a shared mission to uncover the genetic causes of autism and therefore decided to pool our respective resources," said Chinitz of the ongoing collaboration.

A Better Chip?

In the new paper, the authors examined a cohort of 696 unrelated ASD cases using the million-feature Agilent array. The majority of these samples, 615 to be exact, had been previously genotyped with a number of high-density SNP arrays, including Illumina's HumanOmni1 and Omni2.5 BeadChips, and Affymetrix's SNP 6.0 Array and 500K Mapping Set. The authors set out to identify CNVs that had not been picked up in these previous SNP array analyses.

Following PDx's methodology, the CNV data was compared with control data generated on 1,000 healthy individuals using the Agilent chip, allowing the researchers to narrow in on "numerous" rare CNVs in each ASD sample and to identify potential ASD risk genes. "These data allowed us to significantly expand the profile of genetic variants that are potentially causative of ASD and to identify novel molecular pathways affecting ASD vulnerability in this cohort," the authors wrote in the paper.

Interestingly, the million-feature CGH chip also enabled the researchers to identify many variants that earlier studies had missed. Of the 615 ASD cases that had been analyzed on both SNP and CGH arrays, they found that 64 percent of the CNVs were detected exclusively by the Agilent CGH array. The researchers also determined that 75 percent of the CNVs missed by the SNP chips were smaller than 30 kilobases in length.

"Generally speaking, high-resolution SNP arrays were designed to yield binary data on SNPs and were not designed and optimized for copy number detection," said Chinitz of the study results. "Quantitative data is more readily obtained using long oligonucleotide platforms, which typically yield better signal-to-noise ratios," he said, citing a platform comparison paper authored by Scherer and colleagues last year (BAN 6/21/2011).

The authors didn't portray Agilent's chip as being superior for identifying rare CNVs underlying complex diseases like ASD, though. Instead, they stressed that there are "advantages to each microarray platform." In particular, the high-density SNP chips are often more "cost effective" and have the potential to analyze samples at both the SNP genotype and copy number level, while CGH arrays are used in greater frequency in a clinical setting, because of the "better signal-to-noise ratio" achieved in comparison to SNP arrays. "The consensus is that using multiple microarray technologies and prediction algorithms increases CNV detection rates," the authors wrote.

That being said, they did note that the Agilent array is "designed exclusively for CNV detection," relies on a "more uniform probe distribution across the genome as compared with SNP arrays such as the Illumina 1M and Affymetrix 6.0 arrays," is "better at detecting CNVs in segmental duplicated regions compared to SNP arrays," and provides a "better signal-to-noise ratio."

But, as SNP arrays offer researchers the ability to genotype millions of SNPs on array, which current CGH chips, even those that contain SNP content, cannot do, the researchers stuck to their message of platform complementarity.

"Until a single technology [such as] whole-genome sequencing is sufficiently robust to capture most genetic variants, including structural variants such as CNVs, use of multiple platforms will be advantageous," the authors wrote in the paper.

Based on the findings and the state of current genomic research into ASD, the authors concluded with three main points — that given the rarity of some of the risk variants identified, genome-wide scans of tens of thousands of ASD cases will be needed to validate and contextualize the findings; that the results from the study will complement ASD risk factors being identified through current sequencing efforts; and that the availability of the rare CNVs uncovered in the study will serve as an important resource for prioritizing putative ASD risk genes for more in-depth characterization.

A 'Fruitful' Collaboration

In a statement, Scherer credited PDx's technical approach to delineating benign variants from pathogenic ones with the researchers' ability to more "effectively interpret the genome," and called SickKids's ongoing work with PDx a "very fruitful" collaboration.

But the results of the partnership between SickKids and PDx could benefit the diagnostics company too, according to Chinitz.

"Our commercial objective is to develop an early detection pre-symptomatic test for autism within which novel genes will represent a large proportion of the content," Chinitz said. He added that the firm also intends to identify new targets for drug discovery in autism, and has invested "significant resources" to generate high-resolution copy number data on normal individuals in order to be able to differentiate benign from pathogenic variations and generate candidate gene lists.

A number of companies have developed or are developing array- and sequencing-based tests for the early diagnosis of ASD, including Ambry Genetics, Athena Diagnostics, CombiMatrix, GeneDx, Lineagen, SynapDx, and Signature Genomics, in addition to PDx.

Salt Lake City-based Lineagen launched its service, called FirstStepDx, last year. The offering includes genetic testing and personalized counseling, and is run on chromosomal microarrays manufactured by Affymetrix. The Los Angeles-based Center for Autism and Related Disorders began offering the service to its patients and their families earlier this month (BAN 12/4/2012).

Meantime, researchers at the Cleveland Clinic have launched a 600-patient prospective study evaluating a SNP panel developed by French biotech firm IntegraGen to predict autism risk. The researchers are using the Fluidigm EP-1 system to test blood samples in the study(BAN 3/6/2012).

Another firm with an ASD test in development is SynapDx, a Southborough, Mass.-based startup. Last year, SynapDx licensed technology from Children's Hospital Boston to develop a blood-based gene expression test for ASD (BAN 5/3/2011).

Though competition between all these firms will no doubt be intense as more ASD-focused tests become available, Chinitz said his firm has the perspective, and content, to succeed in the developing market.

"When we started the company in 2006 we were relatively alone in appreciating the role of rare variants," Chinitz said. "Today, many others now appreciate the role of rare variants in common disease but what sets us apart is that we actively developed a methodology for discovering them, and we can generate results without having to wait for next-generation sequencing to fully mature."

When asked about what platform the firm would use to deliver its test to market, Chinitz said that it would be a "mistake" to categorize the firm by the tools it uses or the variants it detects. Instead he stressed PDx's ability to use "CNV analysis as a primary discovery tool but proceed to characterize the genes we have discovered for all variant types, including SNVs and indels."

He was also mum on when the firm's ASD test could become available. "A schedule for our development will not be made public and a specific platform will not be defined at this time, suffice to say that it needs to be comprehensive for the detection of all types of genetic variants, not just CNVs," said Chinitz.

But the "important point" is that the test will have content obtained through collaborations with institutions like SickKids.

"Currently, the diagnostic yield of autism tests on the market is between 10 and 15 percent," said Chinitz. "Our test will significantly improve on this figure."

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