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Microarray-based Comparative Genomic Hybridization (aCGH)

By: Aaron Theisen, Ph.D. 2008 Nature Education
Citation: Theisen, A. (2008) Microarray-based comparative genomic hybridization
(aCGH). Nature Education 1(1):45

Move over karyotypesgenetic disorder detection has vastly improved. Researchers are
now using array CGH (aCGH), to quickly scan through an entire genome for imbalances.

Many human genetic disorders result from unbalanced chromosomal

abnormalities, in which there is net gain or loss of genetic material. Traditionally,
cytologists have detected such abnormalities by generating a karyotype of a
person's chromosomes and analyzing the banding patterns therein. Indeed, since
its development in the 1970s, cytogenetic analysis of banding patterns has been
the primary tool for the clinical assessment of patients with a variety of congenital
anomalies. Under ideal conditions, aberrations as small as approximately 5
megabases (Mb) can be detected with banding analysis; such chromosome
rearrangements are termed "microscopic."
In recent years, however, researchers have increasingly turned to newer
cytogenetic techniques. One such method is fluorescence in situ hybridization
(FISH), a technique that uses fluorescently labeled probes to locate the positions
of specific DNA sequences on chromosomes. Yet another popular technique is
comparative genomic hybridization (CGH), which provides an alternative means of
genome-wide screening for copy number variations. First developed to detect
copy number changes in solid tumors, CGH uses two genomes, a test and a
control, which are differentially labeled and competitively hybridized to metaphase
chromosomes. The fluorescent signal intensity of the labeled test DNA relative to
that of the reference DNA can then be linearly plotted across each chromosome,
allowing the identification of copy number changes (Kallioniemi et al., 1992).
Unlike traditional techniques used to detect copy number gains and losses, which
rely on the examination of a single target and prior knowledge of the region under
investigation, CGH can be used to quickly scan an entire genome for imbalances.
In addition, CGH does not require cells that are undergoing division (Speicher et
al., 1993). However, as with earlier cytogenetic methods, the resolution of CGH
has been limited to alterations of approximately 5-10 Mb for most clinical
applications (Lichter et al., 2000; Kirchhoff et al., 1998).

Combining CGH with Microarrays: The Development of Array

CGH
In an attempt to overcome some of the aforementioned limitations associated
with traditional CGH, investigators have developed a newer method that
combines the principles of CGH with the use of microarrays (Schena et al.,
1995). Instead of using metaphase chromosomes, this methodwhich is known
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as array CGH, or simply aCGHuses

slides arrayed with small segments of
DNA as the targets for analysis (Lucito
et al., 2003). These microarrays are
created by the deposit and
immobilization of small amounts of DNA
(known as probes) on a solid support,
such as a glass slide, in an ordered
fashion. Probes vary in size from
oligonucleotides manufactured to
represent areas of interest (2585 base
Figure 1: Diagram of the
pairs) to genomic clones such as
microarray-based comparative
bacterial artificial chromosomes
genomic hybridization (aCGH)
(80,000200,000 base pairs). Because
process.
probes are several orders of magnitude
2008 Nature Education All
smaller than metaphase chromosomes,
rights reserved.
the theoretical resolution of aCGH is
Figure Detail
proportionally higher than that of
traditional CGH. The level of resolution
is determined by considering both probe size and the genomic distance between
DNA probes. For example, a microarray with probes selected from regions
across the genome that are 1 Mb apart will be unable to detect copy number
changes of the intervening sequence.
Regardless of the type of probe, the basic methodology for aCGH analysis is
consistent (Figure 1). First, DNA is extracted from a test sample (e.g., blood,
skin, fetal cells). The test DNA is then labeled with a fluorescent dye of a specific
color, while DNA from a normal control (reference) sample is labeled with a dye
of a different color. The two genomic DNAs, test and reference, are then mixed
together and applied to a microarray. Because the DNAs have been denatured,
they are single strands; thus, when applied to the slide, they attempt to hybridize
with the arrayed single-strand probes. Next, digital imaging systems are used to
capture and quantify the relative fluorescence intensities of the labeled DNA
probes that have hybridized to each target. The fluorescence ratio of the test and
reference hybridization signals is determined at different positions along the
genome, and it provides information on the relative copy number of sequences in
the test genome as compared to the normal genome. The recent sequencing of
the human genome and development of high-throughput methods of robotically
arraying genetic material on a solid surface have enabled the detection of
submicroscopic chromosomal deletions and duplications at an unprecedented
level (DeRisi et al., 1996; Schena et al., 1995; Shaffer et al., 2007).

Advantages of aCGH Technology

The primary advantage of aCGH is the ability to simultaneously detect
aneuploidies, deletions, duplications, and/or amplifications of any locus
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represented on an array; in fact, one assay using this technique is equivalent to

thousands of FISH experiments, with the attendant savings in labor and expense.
In addition, aCGH has proven to be a powerful tool for the detection of
submicroscopic chromosomal abnormalities in individuals with idiopathic mental
retardation and various birth defects. Indeed, several large-scale studies
demonstrate that aCGH has a 10%20% detection rate of chromosomal
abnormalities in children with mental retardation/developmental delay with or
without congenital anomalies; only 3%5% of these abnormalities would be
detectable by other means. For example, in a study of 8,789 cases analyzed by
aCGH, 1,049 (11.9%) had a clinically relevant chromosomal abnormality (Shaffer
et al., 2007).

Studying Specific Chromosomal Regions with aCGH

Because aCGH facilitates simultaneous detection of multiple abnormalities and
offers higher resolution than traditional cytogenetic methods, it has allowed
investigators to focus on various types of rearrangements in particular regions of
chromosomes. In recent years, aCGH has been particularly useful in the study of
subtelomeric and pericentromeric rearrangements.
Subtelomeric Rearrangements
Studies of subtelomeric rearrangements
illustrate how aCGH has revealed an
unprecedented amount of information
about the complexity of the human
genome. Present on all but the short
arms of acrocentric chromosomes 13,
14, 15, 21, and 22, subtelomeric
regions have been the subject of a great
deal of study because they are relatively
gene-rich (Saccone et al., 1992) and
are prone to rearrangement by a
number of mechanisms (Ballif et al.,
2003, 2004). Moreover, rearrangement
of subtelomeric regions has been
suggested to represent a high
proportion of abnormalities in individuals
with idiopathic mental retardation.
The largest study of subtelomeric
abnormalities to date examined 11,688
cases with subtelomeric FISH and
detected pathogenic abnormalities in
2.6% (Ravnan et al., 2006).
Interestingly, recent large-scale

Figure 2: aCGH analysis and

FISH combined can identify
small subtelomeric regions
associated with clinical
phenotypes.
aCGH analysis of the genome of
\"Patient 5\" (a) revealed a deletion
in the subtelomeric region of
chromosome 17. This deletion was
confirmed using FISH in the patient
(b). Chromosome 17 from the
mother and father of the patient is
shown in (c) and (d).
2004 BMJ Publishing Group,
Ltd. Shaw-Smith, C. et al.
Microarray based comparative
genomic hybridisation (arrayCGH) detects submicroscopic
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prospective studies using aCGH on

similar populations show that interstitial
deletions (which are caused by two
breaks in the chromosome arm, the loss
of the intervening segment, and the
rejoining of the chromosome segments)
are two to three times more frequent
than terminal imbalances in subtelomeric
regions (Shaw-Smith et al., 2007).

chromosomal deletions and

duplications in patients with
learning disability/mental
retardation and dysmorphic
features. Journal of Medical
Genetics 41, 241248. All rights
reserved.
Figure Detail

It is important to note that aCGH data can be verified using FISH analysis (Figure
2). For instance, Ballif and others (2007b) recently characterized 169 cases with
subtelomeric abnormalities identified by aCGH. Although the coverage was
sufficient to define the breakpoints in over half (56%) of the subtelomeric
abnormalities, 44% of the abnormalities extended outside the coverage,
suggesting that many such abnormalities are greater than 5 Mb in size. Of these
169 cases, 42 had interstitial deletions. These deletions would have been missed
or incorrectly characterized by subtelomeric FISH panels that use a single clone
to the most distal unique sequence for each region. In addition, six (3.5%) of the
individuals had complex rearrangements that showed deletions along with
duplications or additional deletions. The identification of these sorts of complex
rearrangements suggests that chromosomal abnormalities are often more
complex than previously thought.
Pericentromeric Rearrangements
aCGH has also allowed for the detection of rearrangements in the
pericentromeric regions directly adjacent to the repetitive centromeric regions in
all chromosomes. The pericentromeric regions are known to be prone to
instability because numerous microdeletions, including those causing Williams,
DiGeorge, and Prader-Willi syndromes, occur in these regions. Because of the
high levels of repetitive sequences present in the pericentromeric regions and the
variability in presentation associated with traditional G-banding, rearrangements
in these regions are inherently difficult to assess by chromosome analysis.
However, the recent construction of microarrays targeted to the pericentromeric
regions has allowed for the assessment of copy number imbalances in these
regions. For example, in one study of 8,789 individuals with mental retardation
and/or birth defects (Shaffer et al., 2007), 94 individuals were found to have a
microdeletion in a pericentromeric region, and 42 individuals were found to have
reciprocal duplications in these regions. In addition, 22 individuals had novel
deletions, while 11 individuals had novel duplications of other pericentromeric
regions that were found in two or more patients. Among these individuals were
four with recurrent de novo interstitial deletion in band p11.2p12.2 on the short
arm of chromosome 16. The common clinical features of these patients suggest
that deletion of 16p11.2p12.2 constitutes a novel microdeletion syndrome (Bailif
et al., 2007a). Two other individuals with recurrent interstitial deletions on the long
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arm of chromosome 16 (16q11.2q12.2) were also identified (Bailif et al., 2008b),

and their common clinical features, as well as those of individuals in another
report (Borozdin et al., 2006), suggest that microdeletions of the pericentromeric
region of the long arm of chromosome 16 represent another underappreciated
syndrome.
Many such microdeletion syndromes are caused by nonallelic homologous
recombination (NAHR) mediated by flanking segmental duplications (Shaffer et
al., 2001). This mechanism predicts that reciprocal duplications of these deletions
should occur with equal frequency (Lupski, 1998). However, duplications have
been reported more rarely than expected. One explanation for this finding is that
individuals with duplications usually have milder phenotypes than individuals with
deletions, and these mild phenotypes may not lead to clinical investigation
(Ensenauer et al., 2003; Yobb et al., 2005). Furthermore, duplications involving
segments smaller than 1.5 Mb may be routinely missed even by FISH of
interphase nuclei (Shaffer et al., 1997). However, recent large-population studies
of individuals tested by aCGH have shown that the frequency of reciprocal
duplications is higher than detected in previous studies that used other
cytogenetic technologies (Shaffer et al., 2007; Lu et al., 2007). For example,
duplications of the common Rett syndrome gene MECP2 have been identified in
males with developmental delay (del Gaudio et al., 2006). In addition, the
reciprocal duplications of microdeletion syndromes such as 3q29 microdeletion
syndrome (Ballif et al., 2008a), Williams-Beuren syndrome (Kriek et al., 2006),
and 22q11.21 microdeletion syndrome (Ensenauer et al., 2003) have also been
identified by aCGH. The clinical significance of some of these reciprocal
duplications is not yet known. For instance, only two individuals had de novo
microduplications of 3q29, whereas the remaining cases were inherited from a
carrier parent. Thus, the clinical significance of these duplications is unclear, and
any phenotype may be modulated by an as-yet unidentified genetic modifier.

The Future of aCGH

Array CGH has propelled cytogenetics from the microscope to the computer,
combining CGH with high-throughput microarrays to simultaneously analyze
hundreds or thousands of discrete regions of the genome and identify unbalanced
karyotypes. Array CGH combines the locus-specific nature of FISH with the
global genome view of high-resolution chromosomes; thus, this method
represents the integration of traditional and molecular cytogenetic techniques and
will continue to enable the clinical diagnosis of chromosomal abnormalities at an
unprecedented resolution in the years to come.
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