Comparitive genomic hybridization (CGH) or Chromosomal microarray analysis(CMA) is a modern cytogenetic technique for analyzing variations in DNA copy-number. Alteration in DNA copy number is one of the many ways in which modification of gene expression and function occurs. The technology was initially used for studying chromosomal imbalances in solid tumours.
Large-scale copy number variations in the human genome leading to chromosomal imbalances comprise approximately 12% of the entire genome and some 10% of all known genes [Moechsler 2008].A copy number variation (CNV)is defined as a DNA segment of longer than 1 kb with a variable copy number compared with a normal reference genome. Copy number variations are bounded by a stretch of similar DNA sequences called low-copy repeats or segmental duplications (or interspersed duplication blocks) which may potentially act as recombination hot spots.It should be noted that variations in DNA copy number though associated with disease states, can also be seen in healthy individuals
In recent years chromosomal microarray analysis have come into routine use in child neurology to enable genome-wide evaluation in developmental delay, mental retardation, and autistic spectrum disorders. Chromosomal microarray results should be interpreted with care with the help of published genetic databases,because of the relatively large number of benign CNV in normal individuals. Early consultation should be made with a clinical geneticist.
Microarray technology
- Conventional karyotyping detects chromosomal abnormalities of 5 Mb and larger, including balanced translocations. Even high-resolution chromosome analysis (700-850 bands) would not detect an aberration smaller than 3 Mb.While the diagnostic yield of conventional karyotyping in developmental delay or mental retardation is in the order of 3.7-9.5% by chromosomal microarray analysis the yield has been reported to increase upto 15-20%.
- The DNA samples from a patient and a reference genome are labeled with different fluorescent dyes and are cohybridized to a known genomic sequence. Differences in relative fluorescence intensities of hybridized DNA on the microarray reflect differences in copy number between the genomes of the patient and the reference.
- Bacterial artificial chromosomes (150-200 kb in size) has a higher detection rate than conventional karyotyping, but is unreliable in detecting abnormalities smaller than the bacterial chromosome itself.The coverage of the bacterial artificial chromosome array over the genome is also limited [Bejjani 2005] [Veltman 2006]
- Microarrays based on oligonucleotide probes of various sizes have been developed which can detect variations in copy number of genomic sequence as small as 99 kb [Sebat 2007]
- More advance single-nucleotide polymorphism arrays bypass the need for a labeled reference genome during hybridization and instead compares fluorescence intensities of up to a million loci with those obtained from a series of normal control genomes. Single-nucleotide polymorphism arrays have successfully detected copy number variations (CNVs) of DNA sequences as small as 178 kb [Friedman 2006]. Thus single-nucleotide polymorphisms help in linkage analysis studies adn also allow detection of copy-neutral loss of heterozygosity correlated with uniparental disomy [Altug-Teber 2005] and consanguinity [DeLucca 1991]
- Combining the information of CNV and the minor-allele frequency at the corresponding loci, one can also detect mosaicism in DNA copy number changes, to levels of mosaicism as low as 10-20%.
Overview of array CGH.
(a) Genomic DNA from two cell populations is differentially labeled and hybridized to a microarray. The fluorescent ratios on each array spot are calculated and normalized so that the median log2 ratio is 0.
Plotting of the data for chromosome 9 from pter to qter shows that most elements have a ratio near 0. The two elements nearest pter have a ratio near -1, indicating a reduction by a factor of two in copy number. FISH with a red-labeled probe for the deleted region and a green-labeled control probe (genome locations indicated by the red and green arrows on the ratio profile) shows that the cells contain two copies bound by the green probe and only one bound by the red, consistent with the array CGH analysis7.
(b) Simultaneous comparison of three genomes. Cy3-labeled genomic DNA from melanoma cell line WM-164, Cy5-labeled normal male genomic DNA and fluorescein-labeled normal female genomic DNA were simultaneously hybridized to a BAC array and imaged using a custom-build CCD imaging system (D.P., D.G.A. et al., unpublished data).
The left panel shows the Cy3/Cy5 ratio (cell line/normal male) for each clone plotted in genome order. A large number of aberrations are evident in this nondiploid cell line. The right panel shows the fluorescein/Cy5 ratio (normal female/normal male) with the X chromosome copy-number difference evident. Multigenome hybridizations of this type permit more efficient use of arrays and the inclusion of a control with each unknown specimen.
Unpublished data courtesy of A. Estep (University of California San Francisco, California, USA). source:Nature Genetics 37, S11 - S17 (2005)
