Growing numbers of researchers in the cytogenetics community are now using microarrays that include SNP content, leading some to question the extent to which comparative genomic hybridization arrays will be used in the future.
Cytogeneticists began adopting array-CGH at the beginning of the last decade, progressively moving from bacterial artificial chromosome arrays to two-dye oligonucleotide arrays made by companies like Agilent Technologies and Roche NimbleGen to detect non-polymorphic copy number variation in affected individuals.
Toward the end of the decade, Affymetrix and Illumina entered the market with one-dye SNP arrays, offering researchers the ability to detect polymorphic variations and loss of heterozygosity.
For a time, some labs, such as Baylor College of Medicine's Medical Genetics Laboratory, ran both types of chips, offering array-CGH as a first-tier test and SNP arrays as a second-tier test if the array-CGH results for an individual with an abnormal phenotype came back normal.
Over the past year, though, major array vendors have converged in terms of content, with Agilent and Roche NimbleGen introducing CGH+SNP and CGH/LOH arrays, respectively, and Affy and Illumina adding non-polymorphic probes to their SNP platforms that allow researchers to assess copy number. According to some customers, these kinds of arrays will be the most commonly used going forward, displacing CGH-only arrays.
"The initial problem with SNPs on Affy and Illumina was the quality of the copy number data was not quite as good as the long oligos on the Agilent and NimbleGen platforms [provided], but now Illumina and Affy have added new and higher-quality copy number probes to their SNP arrays, and the copy number companies have added SNP probes to their copy number platforms," said David Ledbetter, chief scientific officer at Geisinger Health System, and current chair of the International Standards for Cytogenomic Arrays Consortium.
"I think that everyone will eventually be using [an array with SNP content] as a primary test," Ledbetter told BioArray News.
Vimla Aggarwal, a researcher in the department of neurology at Columbia University Medical Center, said that her lab has used Affy arrays in the past, starting with the SNP 6.0 and later moving the CytoScan HD, which was launched last year (BAN 7/26/2011). She told BioArray News that arrays with SNP content "give you more information" because SNP arrays can detect uniparental disomy. UPD occurs when an individual inherits two copies of a chromosome, or part of a chromosome, from one parent and no copies from the other, and is thought to be the cause of a number of rare recessive disorders.
SNP arrays can also uncover cases of consanguinity, where the mother and father of an affected individual are closely related, sometimes in the first degree, Aggarwal added.
The issue of uncovering incestuous relationships as part of routine clinical practice has led to discussions about the use of arrays with SNP content and, more specifically, what to do in such situations (BAN 2/15/2011). Lisa Shaffer, chief scientific officer for molecular diagnostics at PerkinElmer and co-founder of its subsidiary Signature Genomic Laboratories, said that the possibility of uncovering consanguinity is "more of a counseling issue," and "not a technological issue."
"The issue is really the patient and physician understanding that SNPs can uncover consanguinity and has that been explained well enough so that the patient or physician is not surprised with a result," Shaffer told BioArray News.
Another question is what counseling will be delivered regarding the regions of homozygosity and the risk of recessive disease, she said. "These issues need to be considered in the pre-test counseling before ordering a SNP array."
According to its website, Signature continues to offer CGH arrays without SNP probes. Shaffer said that Signature is currently validating an array design that includes SNPs that should become available this summer, and that customers will have the option to order arrays with or without SNP content.
Aggarwal agreed that instances of consanguinity are "things that you may not want to find out," but also said that having access to such information "helps if you suspect that it is an autosomal recessive disease" because it allows the researcher to narrow in on a region that might be causing the phenotype.
Aggarwal added that with arrays being increasingly adopted for cancer cytogenetics, chips with SNP content are similarly being favored, because LOH is "very important" in making a diagnosis.
"Learning that a tumor suppressor gene is located in one of those regions of LOH is only going to come out if there is a SNP component on those chips," said Aggarwal.
Still, some users don't think running an array with SNP content is necessary in every situation. Shashikant Kulkarni, medical director of the cytogenomics and molecular pathology lab at Washington University in St. Louis, whose lab has used Affy arrays in the past, said that he is looking into using custom Agilent CGH arrays for cancer applications due to cost constraints.
"In cancer, I cannot justify adding extra cost to a test that is already expensive," Kulkarni told BioArray News. "In those situations, I don't think that a SNP array is the answer," he said. "The better solution is to have a smaller panel of targeted arrays that would be multiplex, rapid, to replace the copy number probes that I am already using" for fluorescence in situ hybridization.
"I can do the smaller, focused, array-CGH-based assays and get some additional information for one-tenth of the cost" of FISH," Kulkarni continued. "People might say that LOH is very important in cancer. Yes it is, I know that, but in some conditions, we don't know that much about LOH regions," he added. "The idea here is not to be in a discovery mode but to be in a clinical diagnostic mode, and I think in that mode, it is very important to keep an eye on cost."
Columbia's Aggarwal has also used CGH-only arrays in recent projects. For example, she and her fellow researchers used an Agilent custom array to study individuals with Glut1 deficiency syndrome, a rare neurological disorder caused by mutations in the SLC2A1 gene. Aggarwal discussed the project during a workshop at the American College of Medical Genetics meeting, held last month in Charlotte, NC.
"It depends on if your hospital or clinic has certain indications that it would like to study," Aggarwal said of using CGH arrays without SNP content. "Glut1 is a big population at our institute, so it just makes sense to have a targeted array with probes to the gene involved to detect duplications and deletions."
In some ways, Agilent is at the center of the debate over using CGH+SNP arrays versus CGH-only or SNP-only arrays. For years the company sold only CGH arrays until it introduced a CGH+SNP product for constitutional cytogenetics research in 2010, followed by a CGH+SNP platform for cancer cytogenetics research last year (BAN 10/11/2011).
Anniek De Witte, Agilent's technical marketing manager for cytogenetics, said the company "expects that its customers will transition to CGH+SNP" arrays from CGH arrays — "especially customers already processing or starting to process prenatal and/or hematological cancer samples."
At the same time, De Witte told BioArray News that researchers currently processing postnatal samples on CGH arrays might not want to revalidate their assays "because the most important data is the copy number data and they perceive the value of being able to also detect copy-neutral aberrations as limited."
"The literature suggests that there are only a very limited number of congenital disorders associated with LOH/UPD," De Witte added. Altogether, she said that Agilent is experiencing a "steady increase in demand" for its CGH+SNP microarrays.
But James Clough, vice president of clinical and genomic solutions at Oxford Gene Technology, said that even though the firm has seen "growing demand" for its CytoSure CGH+SNP arrays for both constitutional and cancer cytogenetics research, he does not expect a decline in CGH array sales in the "immediate future."
"We still see strong demand for our established CGH arrays. The content required by clinicians is well-defined and becoming increasingly stable, although we do still regularly update our designs to include new regions and to improve probe performance," Clough told BioArray News.
He noted that OGT's CGH arrays, which it has sold since 2007, have been validated by customers and "shown to give reliable and consistent results," making users less interested in moving to a new platform.
The location of the lab is also a factor, he said.
"In some geographies, labs still have to implement array-CGH as a front-line test and we are seeing these labs initially use [copy number]-only platforms," Clough noted. "In addition, we are seeing growing interest in using our arrays for prenatal testing, the validation of which is being done on [copy number]-only arrays," he said.
Other factors in platform selection include the focus of the customers' research. Like Agilent, OGT has seen "growing demand" for CGH+SNP arrays both for catalog and custom products, primarily because of the ability to detect LOH at high resolution.
"Higher LOH resolution … is ideal for cytogenetic labs interested in detecting copy neutral changes in consanguineous samples, and we are seeing significant interest in this area," Clough said. Further, in cancer cytogenetics, the availability of SNP probes is "critical" to target LOH regions known to be important predictors of disease progression, he said.
At the same time, Clough acknowledged that the inclusion of SNP probes "does use up some of the real estate on an array."
"If a researcher was primarily interested in [copy-number] detection and they wanted the cost-efficiency of running the 8x60K arrays then the [copy number]-only arrays would be the most attractive," said Clough.
While companies that have offered CGH arrays are adding SNPs to their arrays, firms that have sold SNP genotyping platforms for cytogenetics research are looking to expand the coverage of their arrays, as Affy has done with its CytoScan HD.
"Arrays which only contain SNPs will suffer from gaps in coverage, for measuring copy number changes, since SNPs are biologically not evenly distributed across the genome and are often sparse within genes," said Richard Shippy, Affy's director of strategic product marketing for clinical applications. "Prior DNA-based arrays from Affymetrix didn’t have all genes covered at the resolution we have on CytoScan HD," Shippy told BioArray News.
No Gene Left Behind
As all four major vendors now offer arrays with both copy number and SNP content, previous debates over CGH arrays versus SNP arrays have been subsumed by discussions about the merits of particular vendors' technologies.
Affy is looking to reach customers by touting its high-density arrays. Shippy noted that the CytoScan HD contains 1.9 million non-polymorphic probes and 750,000 SNPs. The firm believes that the "comprehensive" coverage on the array "alleviates the need for customers to continually create new custom arrays since no gene [is] left behind."
Alluding to Agilent, Shippy said that "array manufacturers which rely upon ink jet printing cannot achieve the same high density of probes as seen on CytoScan HD," and therefore "must make choices of which genes to cover as a consequence."
Agilent's De Witte countered that the firm's arrays are "specifically designed for detecting copy number changes" and "offer many advantages compared to SNP platforms originally designed for [genome-wide association studies]."
CGH probes, she continued, allow for "more accurate quantification of copy number changes compared to SNP probes," though she said the addition of SNP content to Agilent's arrays does not affect the performance of the CGH probes.
Finally, De Witte said that the firm's custom capabilities will continue to distinguish it in the market. "With more than 28 million CGH probes to select from, customization will continue to be a key differentiator for Agilent CGH microarrays," she said.
Emily Rorem, director of international product management for clinical markets at Roche NimbleGen, also highlighted the company's custom options. Roche NimbleGen in February launched CGH/LOH arrays on its 3x1.4M and 12x270K platforms, and Rorem said the arrays should be available on its 4.2M and 6x630K platforms before the end of the second quarter.
Roche NimbleGen also launched an ISCA-based catalog design that includes SNP content, but it is only available outside the US, Rorem said. She said that the firm continues to offer its catalog CGX arrays, which do not include SNPs, to researchers, and has no plans to sell the ISCA-based array in the US, citing "excellent uptake and penetration of the CGX arrays in the US market."
However, Rorem noted that Roche NimbleGen has custom CGH/LOH arrays available to US customers interested in SNP content. "Customers provide their own array content, for those [who] require an LOH solution," she said.
Other vendors, including BlueGnome and Illumina, did not respond to questions about the market for chromosomal microarrays in time for this publication.
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