Cytogenetics: from the microscope to the microarray

This feature appeared in the May/June 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.

Associate Professor Howard Slater is in an unusual position for a cytogeneticist: he is the head of the Victorian Clinical Genetics Services (VCGS) cytogenetics laboratory, which functions just like other hospital-based diagnostic labs but just happens to be owned by the Murdoch Children’s Research Institute.

His lab does all of the traditional chromosome testing expected of a diagnostic laboratory, from pre-natal diagnosis to paediatric cancer to infertility in couples, but it also has access to a world-class research facility and to medical doctors, all co-located at the Royal Children’s Hospital in Melbourne.

This co-location allows clinical geneticists to not only work closely with physicians, but to take part in the wonderful research coming out of the Murdoch, particularly for the development of new technologies and new diagnostic tests.

Slater has recently been working with the multinational Affymetrix to help to validate the company’s new range of microarrays, designed specifically for the cytogenetics community. While cytogenetics does use a range of recently developed technologies such as comparative genomic hybridisation (arrayCGH), much of the work is still done by looking down a microscope at chromosomes on a slide.

“That’s a very skilful job and people have to be trained for many years to be able to do it, but no matter how good they are, we still probably only get, in computer terms, 700 or 800 bits of information from that type of analysis,” Slater says.

“So when we look at all of the chromosomes from an individual we can break it down into about 800 bits, and if there are bits missing or relocating then we can spot that. That has been the technology since the early ‘70s.

“The big improvement that has come with these microarrays is that they allow you to look at a couple of million bits of information – from 800 to a couple of million. Now what is happening is that because we can look in such detail the doctors are beginning to not put so much effort into trying to determine themselves what they’ve got, they are saying run it on an array and see if anything comes up. What we are finding is that 15 per cent of those referrals come up with a diagnosis, and that is a huge benefit of these arrays.”

Cytogenetics focus

Affymetrix has just launched two new microarrays specifically for cytogenetics. One is the Whole-Genome 2.7M array, and the other a cytogenetics focused array. The director of clinical market development for Affy, Rich Shippy, says the 2.7M array provides unbiased, whole-genome coverage with 2.7 million markers that measure copy number variations across the genome.

“In addition, that array has 400,000 SNPs,” he says. “We designed markers for every known gene in the genome, so there are greater than 18,000 RefSeq genes, and we decided to put a high density of markers in every one of those more than 18,000 genes. In addition we have a backbone of one marker per kilobase. It’s very gene-centric in its design.”

The focused array, on the other hand, does what the name suggests and focuses on the most well characterised genes. “Outside of those well-characterised gene regions, we have one marker per 16kb,” Shippy says. “It’s a less dense backbone but it’s an evenly spaced backbone. On average, we have one marker per kb on the 2.7M array and on average we have one marker per 10kb on the focused array.”

Of great interest to put-upon lab technicians is that the new arrays do not require the familiar PCR steps or restriction digests. Affy is using longer oligos to achieve this, moving from 25 mer oligos to 49 mer, which greatly simplifies the assay relative to previous products like the SNP 6.0.

What these new arrays will allow cytogeneticists to do is quite startling, Howard Slater says. “In the diagnosis of problems in children, it will increase from two or three per cent to 15 to 20 per cent. That is enormous, so it is not a difficult thing to sell. It’s a staggering increase.

“Importantly it is also probably going to cut out the very few mistakes that are made. With an array analysis, it is all statistically driven, so the computing will basically analyse the data and say this result is abnormal, with a statistical attachment and a probability of being wrong. So suddenly instead of being subjective it is objective and that means that the human error is taken out of it to a large extent and most mistakes are human error.

“It should also make diagnosis quicker, which is very important in pre-natal diagnosis, for example.”

While the SNP information will make it much easier to diagnose potential causes for things like mental retardation and developmental delay in children, also new for cytogeneticists is the ability to look at allelic balance information. The new arrays are able to pick up disorders caused by consanguinities, uniparental disomies and long contiguous stretches of homozygosity (LCSH).

Slater says his lab will still use techniques like arrayCGH and fluorescent in situ hybridisation, but the microarrays will enable a whole new approach.

“Most of what we do is research and development, and because we are using arrays we are finding abnormalities that have never been seen before,” he says. “We are collaborating with people all around the world, by saying to them we have found a new abnormality – have you found one of these? And if you work at it hard enough you usually find that other people have found them as well and then you get together and look at the patient’s features and the genomic abnormality and try to tie the two up.”

This feature appeared in the May/June 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.