Сравнительная геномная гибридизация: метод анализа вариаций числа копий ДНК

Method to evaluate biological samples
Comparative genomic hybridization (CGH) is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions (a portion of a whole chromosome). This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue, and has an improved resolution of 5–10 megabases compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH) which are limited by the resolution of the microscope utilized. This is achieved through the use of competitive fluorescence in situ hybridization. In short, this involves the isolation of DNA from the two sources to be compared, most commonly a test and reference source, independent labelling of each DNA sample with fluorophores (fluorescent molecules) of different colours (usually red and green), denaturation of the DNA so that it is single stranded, and the hybridization of the two resultant samples in a 1:1 ratio to a normal metaphase spread of chromosomes, to which the labelled DNA samples will bind at their locus of origin. Using a fluorescence microscope and computer software, the differentially coloured fluorescent signals are then compared along the length of each chromosome for identification of chromosomal differences between the two sources. A higher intensity of the test sample colour in a specific region of a chromosome indicates the gain of material of that region in the corresponding source sample, while a higher intensity of the reference sample colour indicates the loss of material in the test sample in that specific region. A neutral colour (yellow when the fluorophore labels are red and green) indicates no difference between the two samples in that location. This improved technique allows for the aetiology of known and unknown conditions to be discovered.

The motivation underlying the development of CGH stemmed from the fact that the available forms of cytogenetic analysis at the time (giemsa banding and FISH) were limited in their potential resolution by the microscopes necessary for interpretation of the results they provided. Furthermore, giemsa banding interpretation has the potential to be ambiguous and therefore has lowered reliability, and both techniques require high labour inputs which limits the loci which may be examined. Initially, the widespread use of CGH technology was difficult, as protocols were not uniform and therefore inconsistencies arose, especially due to uncertainties in the interpretation of data. and the image analysis software was made available commercially, which allowed CGH to be utilised all around the world. and Pinkel et al. in 1998 by use of breast cancer cells. This was made possible by the Human Genome Project which generated a library of cloned DNA fragments with known locations throughout the human genome, with these fragments being used as probes on the DNA microarray. Now probes of various origins such as cDNA, genomic PCR products and bacterial artificial chromosomes (BACs) can be used on DNA microarrays which may contain up to 2 million probes.

The DNA on the slide is a reference sample, and is thus obtained from a karyotypically normal man or woman, though it is preferential to use female DNA as they possess two X chromosomes which contain far more genetic information than the male Y chromosome. Phytohaemagglutinin stimulated peripheral blood lymphocytes are used. 1mL of heparinised blood is added to 10ml of culture medium and incubated for 72 hours at 37 °C in an atmosphere of 5% CO2. Colchicine is added to arrest the cells in mitosis, the cells are then harvested and treated with hypotonic potassium chloride and fixed in 3:1 methanol/acetic acid.

Array comparative genomic hybridization (also microarray based comparative genomic hybridization, matrix CGH, array CGH, aCGH) is a molecular cytogenetic technique for the detection of chromosomal copy number changes on a genome wide and high resolution scale. Array CGH compares the patient's genome against a reference genome and identifies differences between the two genomes, and hence locates regions of genomic imbalances in the patient, utilizing the same principles of competitive fluorescence in situ hybridization as traditional CGH. With the introduction of array CGH, the main limitation of conventional CGH, a low resolution, is overcome. In array CGH, the metaphase chromosomes are replaced by cloned DNA fragments (+100–200 kb) of which the exact chromosomal location is known. This allows the detection of aberrations in more detail and, moreover, makes it possible to map the changes directly onto the genomic sequence. Array CGH has proven to be a specific, sensitive, fast and high throughput technique, with considerable advantages compared to other methods used for the analysis of DNA copy number changes making it more amenable to diagnostic applications. Using this method, copy number changes at a level of 5–10 kilobases of DNA sequences can be detected. as of 2006, even high resolution CGH (HR CGH) arrays are accurate to detect structural variations (SV) at resolution of 200 bp. This method allows one to identify new recurrent chromosome changes such as microdeletions and duplications in human conditions such as cancer and birth defects due to chromosome aberrations.

Array CGH is based on the same principle as conventional CGH. In both techniques, DNA from a reference (or control) sample and DNA from a test (or patient) sample are differentially labelled with two different fluorophores and used as probes that are cohybridized competitively onto nucleic acid targets. In conventional CGH, the target is a reference metaphase spread. In array CGH, these targets can be genomic fragments cloned in a variety of vectors (such as BACs or plasmids), cDNAs, or oligonucleotides. Figure 2.

Array CGH has been implemented using a wide variety of techniques. Therefore, some of the advantages and limitations of array CGH are dependent on the technique chosen. The initial approaches used arrays produced from large insert genomic DNA clones, such as BACs. The use of BACs provides sufficient intense signals to detect single copy changes and to locate aberration boundaries accurately. However, initial DNA yields of isolated BAC clones are low and DNA amplification techniques are necessary. These techniques include ligation mediated polymerase chain reaction (PCR), degenerate primer PCR using one or several sets of primers, and rolling circle amplification. Arrays can also be constructed using cDNA. These arrays currently yield a high spatial resolution, but the number of cDNAs is limited by the genes that are encoded on the chromosomes, and their sensitivity is low due to cross hybridization. The latest approach is spotting the arrays with short oligonucleotides. The amount of oligos is almost infinite, and the processing is rapid, cost effective, and easy. Although oligonucleotides do not have the sensitivity to detect single copy changes, averaging of ratios from oligos that map next to each other on the chromosome can compensate for the reduced sensitivity. It is also possible to use arrays which have overlapping probes so that specific breakpoints may be uncovered.

There are two approaches to the design of microarrays for CGH applications: whole genome and targeted. Whole genome arrays are designed to cover the entire human genome. They often include clones that provide an extensive coverage across the genome; and arrays that have contiguous coverage, within the limits of the genome. Whole genome arrays have been constructed mostly for research applications and have proven their outstanding worth in gene discovery. They are also very valuable in screening the genome for DNA gains and losses at an unprecedented resolution. This approach can also be used to study chromosomal aberrations in fetal and neonatal genomes. Furthermore, conventional CGH can be used in detecting chromosomal abnormalities and have been shown to be efficient in diagnosing complex abnormalities associated with human genetic disorders. Some of these changes appear to be common to various kinds of malignant tumors, while others are more tumor specific. For example, gains of chromosomal regions lq, 3q and 8q, as well as losses of 8p, 13q, 16q and 17p, are common to a number of tumor types, such as breast, ovarian, prostate, renal and bladder cancer (Figure. 3). Other alterations, such as 12p and Xp gains in testicular cancer, 13q gain 9q loss in bladder cancer, 14q loss in renal cancer and Xp loss in ovarian cancer are more specific, and might reflect the unique selection forces operating during cancer development in different organs. Several studies have shown that conventional CGH is suitable to detect the deletion, as well as more complex chromosomal alterations. For example, Levy et al. (2002) reported an infant with a cat like cry, the hallmark of CdC, but having an indistinct karyotype. CGH analysis revealed a loss of chromosomal material from 5p15.3 confirming the diagnosis clinically. These results demonstrate that conventional CGH is a reliable technique in detecting structural aberrations and, in specific cases, may be more efficient in diagnosing complex abnormalities. Array CGH may also be applied not only to the discovery of chromosomal abnormalities in cancer, but also to the monitoring of the progression of tumors. Differentiation between metastatic and mild lesions is also possible using FISH once the abnormalities have been identified by array CGH. These small aberrations cannot be detected using cytogenetics or conventional CGH, but can be readily detected using array CGH. As a proof of principle Vissers et al. (2003) constructed a genome wide array with a 1 Mb resolution to screen three patients with known, FISH confirmed microdeletion syndromes, including one with PWS. In all three cases, the abnormalities, ranging from 1.5 to 2.9Mb, were readily identified. Thus, array CGH was demonstrated to be a specific and sensitive approach in detecting submicroscopic aberrations. When using overlapping microarrays, it is also possible to uncover breakpoints involved in chromosomal aberrations.

Though not yet a widely employed technique, the use of array CGH as a tool for preimplantation genetic screening is becoming an increasingly popular concept. It has the potential to detect CNVs and aneuploidy in eggs, sperm or embryos which may contribute to failure of the embryo to successfully implant, miscarriage or conditions such as Down syndrome (trisomy 21). This makes array CGH a promising tool to reduce the incidence of life altering conditions and improve success rates of IVF attempts. The technique involves whole genome amplification from a single cell which is then used in the array CGH method. It may also be used in couples carrying chromosomal translocations such as balanced reciprocal translocations or Robertsonian translocations, which have the potential to cause chromosomal imbalances in their offspring.

A main disadvantage of conventional CGH is its inability to detect structural chromosomal aberrations without copy number changes, such as mosaicism, balanced chromosomal translocations, and inversions. CGH can also only detect gains and losses relative to the ploidy level. In addition, chromosomal regions with short repetitive DNA sequences are highly variable between individuals and can interfere with CGH analysis. Furthermore, the resolution of conventional CGH is a major practical problem that limits its clinical applications. Although CGH has proven to be a useful and reliable technique in the research and diagnostics of both cancer and human genetic disorders, the applications involve only gross abnormalities. Because of the limited resolution of metaphase chromosomes, aberrations smaller than 5–10 Mb cannot be detected using conventional CGH.