The array-based Comparative Genomic Hybridization (CGH-array), known as molecular karyotype, is a revolutionary method to investigate the genome. It was developed to identify DNA anomalies known as Copy Number Variations (CNVs), not detectable by other conventional cytogenetic techniques.
Copy Number Variations are due to the loss of genomic portions (deletions) or to the presence of extra copies of DNA segments (duplications/amplifications).
These DNA abnormalities can cause various diseases such as polymalformative syndromes, mental retardation, autism, epilepsy and cancer.
Over the years, the discovery of new molecular cytogenetic techniques has allowed us to detect structural unbalanced chromosome rearrangements smaller than those evidenced by classical conventional cytogenetics.
The array-CGH analysis of the entire genome guarantees a much higher resolution (100-1000 times) in comparison to other genetic investigations such as the karyotype analysis. It is a valuable tool to detect copy number variations of small size (even a few hundred base pairs). It was useful to discover new microdeletion and microduplication syndromes.
In addition, the array-CGH allows us to define the altered genomic region and genes involved in the rearrangement, improving the understanding of the relationship between the genetic defect and the disease.
The array-CGH analysis is mainly used for postnatal diagnosis of complex phenotypes associated with mental retardation. Molecular karyotype is also used as a second level diagnostic technique in prenatal diagnosis. Limits of this technique in the prenatal field are represented by the inability to identify balanced chromosomal rearrangements and mosaicism with a cell line poorly represented (less than 20%).
Nowadays, microarray technology represents an essential tool in search for a correct diagnosis of several genetic diseases.
Principle of the technique
The principle of CGH-array technique is based on the quantitative comparison between a test DNA and a reference DNA, the latter usually obtained from a healthy subject.
During the analytical process both DNAs are “labelled” by two different fluorescent substances, typically using a red fluorochrome for test DNA and a green fluorochrome for reference DNA. The two labelled DNA are mixed and incubated for several hours onto an array, consisting of a glass or plastic on which short fragments of DNA, known as probes, are fixed. Finally, the platform, after appropriate post-hybridization washing, is subjected to digital image capture by a scanner, able to calculate the intensity of the ratio between the two fluorescent signals.
Each probe represents a specific region of the human genome and the array resolution depends on the number of probes spotted in the array: the higher the number of probes, the more effective the array is in identifying CNVs. By measuring with a specific instrument the intensity of the fluorescence emitted by DNA hybridized onto the array and then comparing them, it is possible to detect copy number alterations of the test DNA.
Currently, there are many types of array-CGH platforms. These can be divided into two main categories, depending on the type of probes fixed onto the array, which are “BAC-arrays” and “oligo-arrays“.
Nowadays, BAC-arrays are considered old generation arrays, since they contain BAC-probes (Bacterial Artificial Chromosome) obtained by cloning fragments of the human genome. Such probes have an average size of about 160,000 base pairs.
The oligo-arrays have probes usually obtained by synthesis with sizes ranging from 20 to 100 bases. The oligo-arrays have smaller and more specific probes, thus represent a significant technological advance in microarray technology.
Numerous scientific publications have pointed out the significant advantages obtained by using oligo-arrays, which guarantee a much higher resolution, greater accuracy in defining the genomic abnormalities and their breakpoints, higher specificity and a significant reduction in false positive and false negative results.
Advantages of the technique
The CGH-array analysis, or molecular karyotype, has many advantages over other conventional cytogenetic techniques, such as conventional karyotype or FISH (Fluorescence In Situ Hybridization).
The conventional karyotype is able to identify several chromosomal numerical and structural abnormalities, but it is limited in its diagnostic capabilities by its resolution. Therefore the conventional karyotype could be normal if the deletion/duplication in the patient with pathological phenotype (patient with mental retardation, autism, epilepsy, etc.) is lower than its resolution (about 10Mb).
The FISH technique is another methodology that allows us to detect rearrangements in specific genomic regions. The FISH is generally used for “targeted investigations” based on a specific clinical indication. This limits the application of this technique as in many cases pathological phenotypes are not related to known syndromes.
Instead the CGH-array analysis allows us to analyse the entire genome in a single test with very high resolution, showing chromosomal abnormalities present in the whole genomic DNA.
The molecular karyotype can be used without a specific clinical diagnosis or a suspected diagnosis, increasing the applicability of this test and the probability of correctly diagnosing pathologies associated with chromosomal abnormalities.
For these reasons, the molecular karyotype is now considered the first genetic test for the diagnosis of genetic syndromes with mental retardation, congenital malformations and neurological disorders.
Compared to other investigation methods, such as the conventional karyotype, the CGH-array has a much higher resolution (100-1000 times), thus allowing the identification of copy number variations of small size, even of a few hundred base pairs, and the subsequent discovery of new microdeletion or microduplication syndromes. Furthermore, the molecular karyotype allows us to define the altered genomic region and thus the genes in it, improving the understanding of the relationship between DNA abnormalities and pathology.
Therefore the array-CGH analysis provides a significant increase in the possibility of making a correct diagnosis.
In addition, the CGH-array analysis, unlike the conventional karyotype, does not need cell cultures: this allows a reduction in reporting time to 3-5 days if the analysis is urgent such as, for example, the molecular karyotype investigation in prenatal diagnosis.