Monday, February 07, 2011

array CGH

When I started as a hematologist, I worked for a guy, who though a hematologist by name, was not much interested in patients with leukemia, sickle cell disease, hemophilia or hemolytic disease. His main interest was in chromosomes. He would take a weekly trip to a hospital for the mentally handicapped to take blood from what he then called FLKs (funny looking kids) in order to recognize strange chromosomal disorders. He is long dead now and I can't find any publications under his name on PubMed, but he got me interested in chromosomes, and I remember a case of CLL who developed MDS with trisomy 8 that I diagnosed while working for him. Of course I am only assuming that it was trisomy 8 because in those days chromosomes could only be distinguished by their size and shape and chromosomes 7-12 all looked the same - they were called 'C'-group chromosomes.

It was not until chromosome banding came into being that we could number the chromosomes accurately. The first type of banding was introduced by Janet Rowley in Chicago, but this required a fluorescent microscope to recognize the quinacrine staining. The much more useful trypsin/Geimsa banding was discovered by my former colleague Marina Seabright shortly afterwards following a laboratory accident when trypsin was spilled onto a chromosome spread. This is now the standard method, but it does suffer from difficulties in spotting very small deletions and additions. This upside down slide shows del 5q, 13q and 20q by trypsin banding.

Readers will be familiar with FISH or fluorescent in situ hybridization, a technique which allows the detection of missing of additional genes, but this will only be of value if you know which gene you are looking for. The picture shows another use, the detection of translocations. Here we see the t(11;14) translocation typical of mantle cell lymphoma. In some of the cells the red and green probes are fused and appear yellow.

There is also spectral karyotyping which is a molecular technique used to simultaneously visualize all the pairs of chromosomes in an organism in different colors. Fluorescently labeled probes for each chromosome are made by labeling chromosome-specific DNA with different fluorophores. Because there are a limited number of spectrally-distinct fluorophores, a combinatorial labeling method is used to generate many different colors.
Spectral differences generated by combinatorial labeling are captured and analyzed by using an interferometer attached to a fluorescence microscope. Image processing software then assigns a pseudo color to each spectrally different combination, allowing the visualization of the individually colored chromosomes. Any translocation, even though quite small can be visualized.

Comparative genomic hybridization (CGH) is the latest method for measuring small cytogenetic aberrations. In a typical CGH measurement, total genomic DNA is isolated from test and reference cell populations, differentially labeled and hybridized to metaphase chromosomes or, more recently, DNA microarrays. The relative hybridization intensity of the test and reference signals at a given location is then (ideally) proportional to the relative copy number of those sequences in the test and reference genomes. If the reference genome is normal, then increases and decreases in the intensity ratio directly indicate DNA copy-number variation in the genome of the test cells.

Data are typically normalized so that the modal ratio for the genome is set to some standard value, typically 1.0 on a linear scale or 0.0 on a logarithmic scale. Additional measurements such as FISH or flow cytometry can be used to determine the copy number associated with a given ratio level. In the picture there is reduplication of the short arm of chromosome 5 plus other fine aberrations which could only have been detected by high resolution CGH such as this.

The latest use of CGH in CLL comes from Harvard where they have compared familial and sporadic CLL. Familial cases had fewer cases with loss at 11q and more cases with a gain of material near the centromere at 14q.


Wayne said...


Given the advantages of both whole genome CGH array and HD Oligo scan used to characterize the MDR will these scans play an important role toward the desired goal of sub-typing the CLL population with the hope for better targeted therapy usage?

Among the shortcomings for CGH are listed the inability to detect point mutations, intragenic deletions, duplications, balanced chromosomal aberrations including Robertsonian translocations, reciprocal translocations, inversions and balanced insertions not to omit mosaicism <30%. That sounds like a lot of categories but how important are they to judge the overall value of the scans?

In the category of balanced translocations, is the coding for correct protein production affected to an important degree in disease progression? Does this also need to be addressed for quality sub-typing?

How much of a weakness for CGH is the need for the reference genome to be normal?

If the clinical utility of CGH at this time is premature in your view, what class of targeted drug therapies will need this class of scanning?

I had better quit but find this topic most interesting.

Hope this finds you coping well.


Terry Hamblin said...

High resolution ACGH is just one more step in the unravelling of the genetic lesions in CLL. It is currently much more expensive than FISH and for looking for the 5 commonest abnormalities - del 11q, del 13q. del 17p, del 6q and trisomy 12, FISH is preferred. CGH is a research tool for seeking moderate sized changes from genomic DNA. It could be used to determine the size of deletions, which may be important for del 13q. SNP analysis will be required for single nucleotide changes. Unsuspected balanced translocations might require spectral karyotyping. These techniques are complementary. Even conventional G-banding is usually sufficient for <30% mosaicism. Epigenetics will be needed to complete the picture. At the moment it is horses for courses.