What’s new in karyotyping? The move towards array comparative genomic hybridisation (CGH)
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- de Ravel, T.J.L., Devriendt, K., Fryns, J. et al. Eur J Pediatr (2007) 166: 637. doi:10.1007/s00431-007-0463-6
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Molecular karyotyping by array comparative genomic hybridisation (array CGH) has doubled the detection rate of pathogenic chromosomal imbalances in patients. This has been possible by increasing the resolution level from the 5 Mb obtained using the conventional karyotype to as low as 100 kb by array technology. Moreover, the technology revealed that over 12% of the human genome includes sub-microscopic benign copy number variable regions. These new findings have implications in genetic counselling and patient management.
KeywordsMolecular karyotypingArray CGHComparative genomic hybridisation
- Array CGH
array comparative genomic hybridisation
bacterial artificial chromosome
fluorescence in situ hybridisation
multiple congenital anomalies
p1 artificial chromosome
single nucleotide polymorphism
Routine chromosome analysis, or karyotyping, has successfully been used for the last 50 years in investigating the cause in patients with mental retardation, specific organ malformations and dysmorphism, whether or not they are part of a syndrome. This has also led to the discovery of genes responsible for various conditions. Standard karyotyping is, however, constrained by the limits of resolution possible by using a microscope. The advent of molecular karyotyping, whereby sub-microscopic copy number changes across the whole genome are evaluated in a single analysis, has greatly increased the detection of pathogenic chromosome imbalances. Whereas standard karyotyping and fluorescence in situ hybridisation (FISH) studies detected chromosome imbalances in 10% of patients with mental retardation, molecular karyotyping has added an additional 10% of detection. This is having a great impact on the understanding of pathologies, the counselling of families and patient management .
Tumour tissue was long-known to have chromosome re-arrangements, especially deletions or duplications (amplifications). In order to detect these regions (and eventually relate them to prognosis), differentially labelled patient DNA and normal reference DNA were simultaneously hybridised to normal metaphase spreads. Regions of loss (deletions) and gain (duplications) were seen as changes in the intensities of the two fluorochromes along the chromosomes. This map of DNA sequence copy number as a function of chromosome location was termed comparative genomic hybridisation (CGH) . As chromosomes are used as the template, the resolution is still only 3–5 Mb nowadays. Solinas-Toldo et al.  and Pinkel et al.  developed array CGH, whereby the hybridisation of the patient DNA takes place on an array of mapped DNA clones instead of metaphase chromosomes. Although chromosomes are no longer visualised under a microscope, the term “molecular karyotyping” is used for this group of techniques since, in analogy to conventional karyotyping, the purpose is the identification of chromosomal imbalances .
The array CGH technology
Selected studies using molecular karyotyping, the selection criteria and results
Vissers et al. 
3 del, 1 pat
2 dup, 1 pat
Shaw-Smith et al. 
7 del, 1 pat
5 dup, 1 pat, 3 mat
Menten et al. 
23 del, 2 pat, 2 mat
7 dup, 2 pat, 1 mat
Rosenberg et al. 
14 del, 1 pat, 1 mat
8 dup, 1 pat, 3 mat
Schoumans et al. 
4 del, de novo
Friedman et al. 
de Vries et al. 
7 del, de novo
3 dup, de novo
Thienpont et al. 
3 del/dup; 1 mosaic
Clinical applications of array CGH
Mental retardation and dysmorphism
Eight studies screening individuals with mental retardation, multiple dysmorphic features and normal traditional karyotypes have demonstrated a high diagnostic yield in MCA/MR patients (Table 1). In summary, at the 1-Mb resolution, 20% to 25% of selected individuals have deletions or duplications or a combination of both. About half of these are conclusively causal for the disorder. The few studies at 100-kb resolution have also detected about 10% of pathogenic interstitial aberrations. The chromosome imbalances occur throughout the genome and are often single cases. In order to correlate the aberrant genotypes with the phenotypes worldwide, collaborative databases of results have been set up, e.g. Decipher (http://www.sanger.ac.uk/PostGenomics/decipher/) and Ecaruca (http://www.ECARUCA.net). Two new microdeletion syndromes have, thus far, been delineated using array CGH. An interstitial microdeletion at chromosome 9q22.3 is associated with a syndrome comprising of macrocephaly, overgrowth, psychomotor retardation and facial dysmorphism . Similarly, a chromosome 17q21.31 microdeletion associated with a common inversion polymorphism results in a new syndrome comprising of moderate mental retardation, marked hypotonia, a long facies, blepharophimosis, ptosis, pear-shaped nose with a broad tip, long columella, large ears and a broad chin [7, 19].
Disruption of dosage-sensitive genes at the translocation breakpoints has long been suspected in patients with mental retardation and/or dysmorphic features. Using array CGH and sequence analyses, both affected patients and normal apparently balanced translocation carrier-parents were shown to have not only insertions and duplications but also disrupted genes at the site of the translocations. More so, with the aid of array CGH, a significant proportion of translocation patients were found to have complex chromosome re-arrangements both in the chromosomes involved in the translocation as well as in other chromosomes [1, 5]. The non-translocation-related chromosome imbalance is, in some cases, responsible for the phenotype.
Array CGH can be carried out on the DNA of single cells, of chorionic villus biopsies and of amniocytes. Due to the complexity in interpreting a complete array (see below), it is foreseen that “targeted arrays” to indicate aneuploidy and known microdeletion/duplication syndromes may be an option in the future .
Investigation of cancer
Both numerical and structural imbalances occur in (pre-)malignant cells and more and more of these are being associated with various prognostic factors. Array CGH has brought a greater number of these to light and is changing the nature of their diagnoses. For example, using CGH, consistent genetic alterations were shown to be associated with primary cutaneous B-cell lymphomas . However, the investigation of cancers is outside the scope of this review.
Research applications of array CGH
Traditional chromosome analysis has led to the identification of disease genes after one or more cases with a specific pathology were found to have the same chromosome translocation-breakpoint or deletion. Since molecular karyotyping now enables the rapid detection of small chromosomal imbalances, gene identification has dramatically increased. Dosage-sensitive genes are now detected on screening numerous patients with a specific pathology and the detection of a patient with a microdeletion/duplication locates the region of the genes involved in the pathology. Using this method, for example, the CHD7 gene responsible for autosomal dominantly inherited (with many de novo cases) CHARGE syndrome was identified (reviewed in ). Likewise, the B3GALTL gene was found to be mutated in patients with the autosomal recessively inherited Peters Plus syndrome . It is likely that the function of many more genes will be identified in this way.
Array CGH is also used at the “full-tiling path” level, where the selected clones overlap, and so, the exact breakpoints of the aberrations can be determined. This is used in the known microdeletion/duplication syndromes which involve multiple genes. The purpose here is to correlate the various components of the phenotype with the loci/genes within the affected chromosomal region. For example, array CGH and FISH analysis permitted the delineation of the 2q32.2q33 syndrome in four patients, which were then compared to a further nine patients. All of these cases shared a minimal deleted chromosomal region and striking phenotypic similarities. As all patients had a cleft or high palate, it was speculated that hemizygosity of the SATB2 gene within this region may be the underlying cause .
Challenges in interpretation
Copy number variation/polymorphisms
Numerous regions with non-pathogenic variations in the number of DNA copies (more or less than two copies) are scattered throughout the human genome. Using both array CGH and single nucleotide polymorphism (SNP) genotyping arrays on the 270 individuals of the HapMap collection from ancestry in Europe, Africa and Asia, 1,447 sub-microscopic copy variable regions in the human genome were found . This involves at least 12% of the genome and includes hundreds of genes in deletions, duplications, insertions and complex multi-site variants. Interestingly, population-specific copy number variations have been detected, which needs to be considered when analysing the results of patients. This is facilitated by access to the Database of Genomic Variants (http://projects.tcag.ca/variation/) and also the results of the HapMap collection mentioned above , both of which are visualised in Ensembl (http://www.ensembl.org/).
If the imbalance is familial and not a known benign copy number variation, the phenotypic relationship is difficult to interpret
If the aberration in a patient involves a known microdeletion/duplication syndrome, the imbalance is considered as pathogenic
If the imbalance has occurred de novo in the patient, and especially if it contains genes with effects compatible with the clinical findings of the patient, this is in support of its pathogenicity but is not absolute proof
In conclusion, the pathogenicity of a chromosomal imbalance in a patient needs to be proved in order to be of use in the management of the patient and in counselling the family as to the implications . To this end, the collaborative efforts through international databases such as Decipher and Ecaruca will, hopefully, with time, permit the detection of similar cases and the determination of the pathogenicity of the individual aberrations.
Thus, when investigating a patient using molecular karyotyping, investigation of the parents and additional family members may often be necessary in order to interpret the results. Without the availability of DNA from parents, molecular karyotyping at the higher (100-kb) resolution is not possible, as the hundreds of polymorphisms may be difficult to interpret.
The introduction of molecular karyotyping has doubled the detection rate of chromosomal imbalances in patients with mental retardation and multiple congenital anomalies or dysmorphism and is, therefore, rapidly being introduced as a routine diagnostic technique in genetic diagnostic centres. It is, however, important to carefully select patients to undergo this investigation, as mutations in single-gene disorders will not be detected. Also, the technology is advancing the gene detection rate at a faster pace. The understanding of copy number variations in the human genome is now better understood than ever and its implications in diagnosis and the implications in genetic counselling are being rapidly uncovered. This is challenging the sector into re-thinking the indications for traditional chromosome analysis as opposed to molecular karyotyping.