Comparative Genomic Hybridization (CGH)

Comparative genomic hybridization (CGH)

Comparative genomic hybridization (CGH) or Chromosomal Microarray Analysis (CMA) is a molecular-cytogenetic method for the analysis of copy number changes (gains/losses) in the DNA content of a given subject's DNA and often in tumor cells. CGH will detect only unbalanced chromosomal changes. Structural chromosome aberrations such as balanced reciprocal translocations or inversions can not be detected, as they do not change the copy number. CGH is based on co-hybridization of two differentially labeled genomic DNAs (eg. tumor and normal) to human metaphase chromosome spreads.

Comparative genomic hybridization (CGH)

It is based on the co-hybridization of differentially labelled test and reference DNA onto metaphase spreads, which usually have been prepared from peripheral blood lymphocytes of a healthy donor. The signal intensity ratios of the two labels along the chromosomes then reflect DNA copy number changes in the test genome relative to the reference genome. Its resolution is limited to about 3-10Mb

 Comparative genomic hybridization (CGH) is a fairly new molecular cytogenetic technique that allows detection of DNA sequence copy number changes throughout the genome in a single hybridization. CGH is based on co-hybridization of differentially labeled tumor and normal DNA to human metaphase chromosomes.

Illustration of a comparative genome hybridization (CGH) experiment that allows the detection of chromosome imbalances in tumor DNA. Tumor DNA and control DNA are labeled with different fluorochromes and hybridized to normal diploid metaphase chromosomes. The relative hybridization intensities of the tumor and control DNA are measured along each chromosome. More intense fluorescence of the tumor DNA compared to the control DNA indicates gain of chromosomal material in the tumor sample (gain of entire chromosomes or chromosome fragments, labeled green in the idiogram). Less intensive signals indicate loss of chromosomal material in the tumor sample (red in the idiogram).

After extraction of test DNA (i.e. from a tumor sample) and normal DNA (i.e. from peripheral blood), the samples are differentially labeled with discernable fluorochromes (i.e. tumor DNA with FITC [green] and control DNA with TRITC [red]) (Figure 3A). The two genomes are combined with an excess of human Cot-1 DNA and then hybridized to normal metaphase chromosomes (Figure 3B). Images of metaphase spreads are then acquired with a (charged coupled device) CCD camera and fluorochrome-specific optical filter sets to capture the FITC and TRITC fluorescence (Figure 3C). Differences in fluorescence intensity values between tumor and control DNA represent gains and losses of specific chromosomes or chromosomal regions (du Manoir et al., 1995). For example, a gain of a chromosomal region in the test sample would result in an increased intensity of green fluorescence. A loss within a chromosomal region in the tumor would be indicated by a shift towards red intensities. Specialized CGH analysis software measures fluorescence intensity values along the length of the chromosomes and translates the ratios into chromosome profiles (Piper et al., 1995). The ratio of green to red fluorescence values is used to quantitate genetic imbalances in tumor samples

Tumor DNA

Normal DNA

After Hybridization

Figure : Schematic representation of Comparative Genomic Hybridization.

(A) CGH begins with the isolation of both (1) genomic tumor DNA and (2) DNA from an individual with a normal karyotype (reference or control DNA). The two genomes are differentially labeled such that, for instance, the tumor DNA can be detected with a green fluorochrome (FITC) and the control DNA with a red fluorochrome (TRITC). (3) The differentially labeled genomes are then combined in the presence of excess Cot-1 DNA. (B) Both the probe and karyotypically normal target metaphase chromosomes are heat denatured prior to hybridization for a 24-72 hour period at 37C. (C) Following a series of detection steps, metaphase chromosomes are imaged by epifluorescence microscopy with DAPI, FITC and TRITC filters consecutively. (1) The differences in fluorescence intensities along a chromosome are a reflection of the actual copy number changes in the tumor genome relative to the normal reference. The result of the hybridization shows gains and losses; in the event that a specific chromosome region is lost in the tumor, the color of that region is shifted to red. A gain would be represented by an increased intensity of the green fluorescence. (2) A minimum of 5 metaphases (or 10 copies of each chromosome) are analyzed to determine an average ratio profile. A ratio of 1 represents an equal copy number in the tumor and the reference genome. The vertical lines to the left and right of the chromosome represent a loss (< 0.8) and a gain (>1.2), respectively.

CGH
The intensity ratio of the two fluorescence signals gives a measure for the copy number ratio between the two genomic DNA samples. CGH can be either performed on metaphase chromosomes or using BAC DNA as array-CGH.

Comparative genomic hybridization (CGH) ratio profiles illustrating loss of chromosome 6q in cases 3, 4, 5, 6, 7 and 8. The averaged CGH ratio profile shows primary central nervous system lymphoma (PCNSL) DNA versus normal control DNA (blue line). The vertical red and green bars right of the ideogram indicate the cutoff levels for gains (1.15>) and losses (0.85<)

Fig.. Flowchart for CGH analysis. First, equal amounts (500 ng) of tumor DNA is labeled with green fluorochrome-conjugated nucleotide, such as Spectrum greendUTP or fluorescence isothiocyanatedUTP (FITCdUTP) by nick translation, whereas reference DNA from peripheral lymphocytes of normal male or female donors is labeled with red fluorochrome-conjugated nucleotide, such as Spectrum reddUTP or Texas reddUTP. The sizes of the probes are optimized to a range from 500 bp to 2 kb. The labeled DNA (200 ng) is mixed with 10 g of unlabeled human Cot-1 DNA for blocking ubiquitous repetitive sequences commonly detected on the centromeric and heterochromatic regions; then, it is ethanol precipitated and redissolved in 10 l hybridization solution. The resulting probe mixture is denatured at 76C for 7 min and reannealed at 37C for 1 h. Metaphase chromosomes are also denatured at 76C in 70% formamide solution for 34 min. The denatured probe mixture is dropped onto the metaphase chromosome slide, covered with a cover slip, sealed with rubber cement, and then allowed to hybridize by incubating the slide in a moist chamber at 37C for 3 d. After hybridization, the slides are washed twice with 50% formamide with 2 SSC at 43C for 10 min, followed by three changes with PN buffer at room temperature, 10 min each for removing the unbound probe. The slides are counterstained with diamidino-2- phenylindole (DAPI) and mounted in Vectashield mounting medium. Fluorescence signals from the hybridized chromosomes are captured and analyzed using CGH analysis software, such as the QUIPS XL Genetics Workstation system. Typically, 610 sets of metaphase chromosomes from one sample slide are observed under a fluorescence microscope equipped with a cooled CCD camera. The filter system consists of a triple-bandpass beam splitter, a triple-bandpass emission filter, and three single-bandpass excitation filters mounted on a computer-controlled filter wheel. This design allows collection of sequential, properly registered images from three fluorescence channels. The ratio profile of green (tumor) to red (control) fluorescence intensity is plotted as a function of locations along individual chromosomes using software. Data from 610 captured metaphases are pooled to obtain an averaged ratio profile for each sample (or patient). Genomic aberrations, such as gains (amplification) or losses (deletion), at certain chromosome regions are defined by setting the thresholds at 1.2 and 0.80, respectively.

CGH Methodology
DNA from subject tissue (tumor) and from normal control tissue (reference) is labeled with different colors. Tumor DNA is labeled with a green fluorescent fluorophore (FITC, fluorescein isothiocyanate) and normal DNA is labeled with rhodamine/Texas red, which fluoresces red After mixing subject and reference DNA along with unlabeled human cot-1 DNA (placental DNA that is enriched for repetitive DNA sequences such as the Alu and Kpn family) to suppress repetitive DNA sequences, the mix is hybridized to normal metaphase chromosomes or, for array- or matrix-CGH, to a slide containing hundreds or thousands of defined DNA probes. Images of metaphase spreads are then acquired with a (charged coupled device) CCD camera and fluorochrome-specific optical filter sets to capture the FITC and rhodamine fluorescence.

CGH Methodology
Differences in fluorescence intensity values between tumor and control DNA represent gains and losses of specific chromosomes or chromosomal regions. For eg.,
a gain of a chromosomal region in the test sample would result in an increased intensity of green fluorescence. A loss within a chromosomal region in the tumor would be indicated by a shift towards red intensities.

Specialized CGH analysis software measures fluorescence intensity values along the length of the chromosomes and translates the ratios into chromosome profiles. The ratio of green to red fluorescence values is used to quantitate genetic imbalances in tumor samples.

CGH
Chromosomal CGH is capable of detecting loss, gain and amplification of the copy number at the levels of chromosomes. The sensitivity of CGH in detecting gains and losses of DNA sequences is approximately 2-20 Mb. For example, a 10-fold amplification of a 200 kb region should be detectable under optimal hybridization conditions. Detect a single copy loss the region must be at least 5-10 Mb in length.
Detection of amplifications (e.g. tens or hundreds of copies of one or few neighboring genes) is known to be sensitive down to less than 1 Mb

Applications of CGH
CGH has been used to detect genetic aberrations in a variety of malignancies: It has already revealed a complex genetic nature of cancer and indicated several novel chromosomal regions that are frequently altered in tumors. Several CGH studies on prostate cancer have been published, e.g., loss of 6q, 8p, 13q, and 16q, as well as gains of 7p, 7q, 8q and Xq, have been found to be common alterations in prostate cancer. CGH findings led also to the discovery of androgen receptor (AR) gene amplification in hormone-refractory recurrent prostate carcinomas.

Array Comparative Genomic Hybridization

Also CMA, Chromosomal Microarray Analysis, Microarraybased comparative genomic hybridization, array CGH, a-CGH, aCGH, is a technique to detect genomic copy number variations at a higher resolution level than chromosomebased comparative genomic hybridization (CGH). Combination of Microarray techniques and CGH A grid of well-defined, genomically mapped DNA fragments (e.g. BAC clones, cDNA, oligonucleotides) immobilised on a glass surface replace condensed metaphase chromosomes as the hybridisation target .

Fig. Array Comparative Genomic Hybridization

aCGH
The genome to be tested and a normal genome as reference are used as probes. They are differently labeled. In the example shown here, the DNA from an individual to be tested is labeled with the fluorophore Cy5, which induces green fluorescence. The DNA from another individual as control is labeled with Cy3, which induces red fluorescence. If the amount of DNA is the same, a yellow signal will result. However, wherever there is an imbalance, the signal will be red if the amount of DNA is reduced due to a deletion and green in case of duplication.

aCGH
The resolution achieved can be dramatically increased, since it depends on the number of gene fragments represented and their separation distance. Using this method, copy number changes at a level of 5-10 kilobases of DNA sequences can be detected. These high resolutions permit the detection of sub-microscopic genomic imbalances, as well as the precise breakpoint determination for genomic aberrations. Therefore, array-CGH can complement the standard cytogenetic methods (chromosome analysis, subtelomere analysis, FISH) in prenatal, postnatal and tumour-cytogenetic analysis. In addition, array-CGH enables the identification of chromosomal segments, showing copy number aberrations (deletion, duplication or amplification). Array CGH is a fast and efficient means of detecting deletions and duplications on a genome-wide scale.