The recent availability of higher-resolution microarray platforms has spurred more cytogenetics laboratories to perform genome-wide, rather than targeted scans, of samples to help them identify chromosomal abnormalities.
The result has increased the labs' rates of detecting abnormalities, according to sources at some of the US' busiest cyto labs.
In separate interviews, Baylor College of Medicine's James Lupski, Signature Genomic Laboratories' Bassem Bejjani, and the Children's Hospital of Philadelphia's Nancy Spinner recently discussed how the new arrays, sold by Agilent Technologies, Affymetrix, and Illumina, have affected their services.
Some common trends include a convergence to whole-genome arrays from the targeted arrays that were used earlier by cytogeneticists; the dominance of oligonucleotide-based array platforms in cyto labs that once relied heavily on bacterial artificial chromosome arrays; a rise in detection rate due to the availability of higher-resolution platforms; and growing archives of cases that inform decision making within cyto labs and could eventually be used to design better platforms.
Baylor College of Medicine's Medical Genetics Laboratories in Houston is one of the major centers offering array-based genetic testing in the US. According to Lupski, a professor of molecular and human genetics and pediatrics at BCM, MGL has run more than 21,000 arrays on samples through its chromosomal microarray-analysis and prenatal-testing services since it began offering CMA in 2004.
Baylor initially used a BAC-based platform that was eventually commercialized by Spectral Genomics, which was acquired by PerkinElmer in 2006. In 2007, Baylor switched to an oligo-based CGH platform manufactured by Agilent to take advantage of the higher resolution provided by the chips (see BAN 4/3/2007).
Baylor is currently using 105,000-probe Agilent-manufactured chips in its service, though it also uses Affymetrix's Cytogenetics Solution, based on its GeneChip SNP 6.0 Array, in its National Institutes of Health-sponsored prenatal studies. Lupski said that Baylor will soon upgrade to using 180K Agilent arrays, taking advantage of the firm's recent density upgrade to a million-feature format, which launched earlier this month (see BAN 3/3/2009).
Lupski spoke with BioArray News at the Wellcome Trust Sanger Centre-hosted Genomic Disorders conference in Cambridge, UK, earlier this month.
Baylor's journey from BAC arrays to high-resolution oligo arrays has gone hand in hand with the availability of new technology, Lupski said. "Initially, [the arrays] were targeted based on clinically relevant regions," Lupski said. "Then [they] were targeted based on the BAC-emulated oligo" Baylor placed on the first generation of its Agilent-manufactured platform.
"Now, as there was more and more coverage, particularly as we switched to the 105,000 oligos, [we approached] a genome-wide density for the ‘backbone,’" Lupski said.
This higher resolution view of the genome has had several effects on Baylor's service, including a better understanding of genetic abnormalities; an increased ability to make a call on a sample; and a growing archive of data that can be used to make future decisions about abnormalities in a sample, he said.
"What we have found along the way is that our initial conception of a lot of the genomic disorders is that they were microdeletion syndromes below the resolution level" of traditional cytogenetic technologies like molecular karyotyping or fluorescence in situ hybridization, Lupski said.
"As we have better analysis of the genome in the context of a clinical situation, we are finding that there are many rearrangements that have evaded the resolution capabilities of BACs," he said. "In [traditional] cytogenetics, you are lucky if you see a 5 megabase deletion. BACs got us down to a couple hundred kb. Oligos got us down to 50 to 70 kb. Higher-density oligos will enable detection of smaller and smaller-sized rearrangements."
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As the resolution of Baylor's arrays has increased, so has the likelihood of identifying a copy number variant in an assay that a clinical cytogeneticist will deem pathologically significant, Lupski said.
"We've gone from 6 percent to 15 to 20 percent," he said. "A 15 to 20 percent rate of detecting a submicroscopic CNV that may be of clinical relevance is unbelievable if you think that in your average chromosome study you were lucky to find an abnormality a few percent of the time in the referred cases," he added.
With more than 21,000 arrays run, Baylor has also had to develop tools to archive and retrieve the data from past cases. Lupski said that all information is kept in an internal database that benefits both Baylor and referring physicians.
"When you find a new copy number variation [for which] you don't know the significance, you go to the database to see how many times you have seen it before and to answer other questions," Lupski said. "Within the laboratory, that information can be very helpful. It's questionable as to how useful that information could be in the outside world. But we've been now offering our service for five full years. So we have some real experience here."
Like other cyto labs, Baylor is also being targeted by a number of companies competing for market share. Beyond Agilent and Affy are companies like Illumina, Roche NimbleGen, Oxford Gene Technology, BlueGnome, Empire Genomics, and others who are looking to cash in on what they see as a growing market opportunity.
"Every time new platforms come along, we side by side compare them on usually at least 100 control individuals," Lupski said. "But they have to show us real data and that they know what they are talking about. We won't fall for a sales pitch."
Expanding in Spokane
At Signature Genomic Laboratories, the adoption of array technology by cytogeneticists is fueling the physical growth of the six-year-old company. In December, Signature leased 4,600 square feet of office space in downtown Spokane, Wash., to house its finance, sales, marketing, business-development, and information-technology departments (see BAN 12/16/2008). Last May, Signature completed construction of an 18,000-square-foot space for its headquarters and laboratory, also located in Spokane (see BAN 6/3/2008).
"The transition of traditional cytogenetics to arrays is accelerating," said Chief Medical Officer Bassem Bejjani. "The whole market is expanding and we are also growing our market share. Recently we got our New York [clinical laboratory permit]. That really increases the number of patients that we are able to serve."
Bejjani also spoke with BioArray News at the Genomic Disorders meeting. According to Bejjani, Signature has run 35,000 cases to date, many of which were run on its dual array platforms, the BAC-based SignatureChipWG, and the Agilent-manufactured, oligo-based SignatureChipOS, both of which the firm debuted at the end of 2007 (see BAN 10/23/2007).
Bejjani said that the availability of higher-resolution arrays has increased Signature's detection rate, so that it now has a detection rate of clinically significant abnormalities that is around 20 percent. At the same time, the new arrays have also increased the number of CNVs detected that are of unclear clinical significance. "Those are more challenging to interpret. And this is the reason we have developed more algorithms and software tools to help us understand the significance of these variations," Bejjani said.
The challenge of interpreting the clinical significance of variation is another effect of high-resolution arrays on the cytogenetics field. Each month, dozens of papers are published that provide some insight into particular variants. But how does a lab like Signature integrate those findings into a clinical service?
"It depends on the paper," Bejjani said. Consider the 1q21 deletion, "where we know now that the deletion causes microcephaly and mental retardation. For a long time, we were calling these as being of unclear clinical significance until the paper in Nature Genetics came out that showed clearly in a large cohort that these individuals have mental retardation and microcephaly.
"However, there are some people with this deletion who are shown to have mental retardation and others who are normal," said Bejjani. "So we give this information in the report and we have the clinician determine whether the abnormality we found correlates with the clinical presentation."
Higher-resolution arrays have changed Signature's service in one other way: The multiplexing capability of Agilent's platform allows the firm to save money. "You can put more than one patient on a slide or array," said Bejjani. "You have more subarrays and therefore you can multiplex and bring down the cost of the analyses, to make the service accessible to more people."
Still, he said that Signature's R&D department is constantly reviewing new platforms as they come onto the market. He said that Signature is currently working with the Affy, Agilent, and Roche NimbleGen platforms in R&D.
"All of these companies keep coming out with new platforms and we play with them," Bejjani said.
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Opting for SNPs
Though it has run fewer samples, the cytogenetics lab at CHOP can be distinguished from Baylor and Signature in that it has opted to use a SNP genotyping platform with CNV content rather than a CGH platform in its services.
The lab, directed by Nancy Spinner, began offering array-based chromosomal analysis on Illumina's 610-Quad BeadChip in May 2008, partially due to CHOP's previous experience of using the BeadChip platform in R&D.
"We think that the combination of the genotyping data and the intensity data is very powerful," Spinner told BioArray News this week. "We can see an increased frequency of mosaics; we are able to diagnose uniparental disomy," she said. "You can look at how things are inherited in one fell swoop from genotyping data, and you can look at mechanisms and timing of nondisjunction in meiosis and mitosis by evaluation of the genotype."
Prior to implementing Illumina, Spinner's lab used standard cytogenetics, though it did outsource a subset of samples to Signature due to demand for array-based testing. "I think everyone agrees that the use of array platforms has revolutionized cytogenetics," Spinner said. "When we brought them in house we knew what was coming, but it has increased the diagnoses we are able to make, it gives up fairly precise breakpoints, so we know what genes are involved in deletions and duplications. We are finding more subtle abnormalities and mosaics than we had seen previously."
Since May 2008, Spinner's lab has performed more than 1,600 cases and it is now running over 200 samples a month. Like Baylor and Signature, the data provided by the higher-resolution arrays have encouraged CHOP to develop a process to establish clinical significance in a previously unknown variation.
Spinner said that CHOP uses a "complicated algorithm" to assess whether a particular variant is causing a particular phenotype. To provide this kind of service, the cyto lab at CHOP relies on in-house resources, such as CHOP's genotyping center and its bioinformatics group.
Spinner's lab also has the sample data collected in an in-lab database to inform future samples and is considering "streamlining the processes and what we want to see in the arrays in the future." According to Spinner, the accumulation of data at CHOP as well as in other cyto labs will inevitably guide future generations of chips for cytogenetic testing.
"The competition is leading to leapfrogging from one platform to the next and they are getting better and better," Spinner said about the arrays on the market. "We have a long way to go to a perfect array, but we will get to a point where we can diagnose all significant deletions and duplications on one platform," she said.
What will that perfect array look like? According to Spinner, the perfect platform will target all known dosage-sensitive genes at the exon level. "As we get more comfortable with what those genes are we want to target every gene that we know to be dosage sensitive," Spinner said. "The data on these genes is increasing and we want to be able to use that," she said. "Once a dosage map of the human genome is established, every cytogeneticist can use that map to make their calls."