Array CGH enters the realm of clinical diagnostics

Array-based comparative genomic hybridisation (aCGH) is helping researchers to track down smaller and smaller DNA copy number variations associated with a wide range of diseases, from developmental disorders to cancer.

For the cytogeneticist, it is set to take over from karyotyping and fluorescence in situ hybridisation (FISH), until now the conventional methods used in cytogenetics. What aCGH will offer in the near future is a much higher sensitivity than these methods while keeping the price of testing to manageable levels.

Dr Swaroop Aradhya is a research fellow at the clinical cytogenetics lab at Stanford University Hospital in California. He was in Australia recently to speak to guests of Agilent Technologies about a study his group has conducted using CGH in a diagnostics context. "We have been interested in using CGH as a diagnostic tool to study our patients, particularly where we suspect there is a genetic component but conventional cytogenetic methods are inadequate," Aradhya said.

"What we really needed in a diagnostics context is something that offers superior resolution and a whole genome view. This is where array CGH comes in. We have tested a panel of 20 patients with developmental delay, some also with mental retardation and dysmorphic features." Aradhya said genetic alterations were the most commonly identifiable cause of developmental delay in children, but it was often difficult to identify the specific alteration, especially if the patient presents a mild phenotype.

His group of 20 patients had all had conventional cytogenetic tests - karyotyping, FISH and in some cases subtelomere-targeted FISH. "In studying these patients we also wanted to compare two commercially available array CGH platforms - one based on bacterial artificial chromosomes (BAC) and the other one on oligonucleotides.

"We started our analysis with a one megabase resolution whole genome BAC array. One of our patients had development delay and a bleeding disorder. We discovered a large deletion on the X chromosome in this patient. Based on the positions of the BACs that identified this deletion, we estimated the mutation to be about 4 to 7 Mb in the Xq26-Xq27 region, an interval that contains the Factor IX gene.

"Another patient had developmental delay and a host of dysmorphic features - his disorder is generally known as the Nablus mask-like facial syndrome. The BAC arrays showed a deletion of about 3 to 6 Mb in 8q22 based on the positions of the BAC clones on the genome physical map.

"We tested the same group of 20 patients on another platform, the 44k Agilent whole genome oligonucleotide array. With the BAC arrays, because they are spaced at about 1mb apart, it is hard to get a really good delineation of the genetic alteration. But because the oligonucleotide array has a much higher density, with the patient with the bleeding disorder we could narrow it to a 4.9mb deletion."

The BAC arrays picked up six different alterations: five deletions and one duplication. The oligo arrays identified all of those alterations and an extra four.

He said the higher resolution of oligo arrays allowed the researchers to pick up smaller mutations that the BAC arrays missed. "There were 10 mutations identified in total, of which nine were deletions and one a duplication, ranging from 208kb to 8.3mb. Most of these mutations are new and only a few have been reported before."

He believes that array CGH will increasingly become the technology of choice for cytogeneticists, with high-resolution karyotyping and subtelomere FISH eventually becoming obsolete.

"These are tests that have a low yield as it is," he said. "Array CGH has a much higher sensitivity. I hear that physicians are starting to ask for aCGH first now, but I think that until we appreciate the technology fully we will probably do both aCGH and karyotyping in the foreseeable future. "There is also talk of it being used in cancer diagnostics as array CGH can detect loss or gain of DNA, but cancers have various inversions and translocations that won't be picked up by array CGH."

Aradhya said Standford Hospital hoped to set up a diagnostic service using the technology later this year.

Agilent Technologies' applications scientist Dr Russell McInnes agreed that CGH was being widely adopted by the genetics community, particularly in cytogenetics.

"The basic principle is to look for DNA copy changes across the genome," McInnes said. "With oligo arrays we can do this at high resolution, high throughput and it's a quantitative method.

"Researchers have the flexibility to use a catalogue chip that we have designed to do whole genome coverage or use our eArray facility to make a custom design at no upfront cost to address specific regions that you want to interrogate at high resolution. The assay is very simple." He said Agilent had decided to use 60 mer oligonucleotides rather than the 25 mer used by other products based on empirical testing. "60 mer is typically 10 more sensitive than the 25 mer with our platform," he said.

"We have CGH catalogues for human, mouse and rat and they give you the ability to do genome-wide surveys. The 244K array has about 8.9kb median resolution and the 105K has 21kb median resolution.

"We make intelligent choices about the probes that we put on the array so we have targeted about 70 per cent of our probes towards the gene-rich regions. 97.5 per cent of the genes have more than four probes on them in the 244K array. The other 30 per cent is spread around the genome with good coverage of telomeres, so we can pick things up that other technologies don't, particularly SNP-based technologies.

"Our protocol is quite straightforward - we can go down to 500 nanograms without amplification or the standard protocol starts at 100 nanograms of total genomic DNA with amplification.

"CGH analytics can also do joint analysis of gene expression and CGH data. For instance, if you run a sample on Agilent gene expression chips and also on aCGH chips you can import that into CGH analytics and do some statistics looking for relationships between amplification and increases in expression or copy number deletion and decreases in expression."

McInnes said the formats available for custom arrays are 244K, 2 x 110K and 4 x 44K, or 44K, 22K or 2 x 11K. "In terms of the performance between the 44 and the 244 - the 44 does a very good job of picking things up that other technologies don't, but the 244 does even better."