Comparative Hzbridiyation

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10.1146/annurev.genom.6.080604.162140
Annu. Rev. Genomics Hum. Genet. 2005. 6:331–54

doi: 10.1146/annurev.genom.6.080604.162140
Copyright c 2005 by Annual Reviews. All rights reserved
First published online as a Review in Advance on May 25, 2005
COMPARATIVE GENOMIC HYBRIDIZATION

Daniel Pinkel1 and Donna G. Albertson1,2
Comprehensive Cancer Center, Department of Laboratory Medicine,1 Cancer Research
Institute,2 University of California, San Francisco, California 94143;
by Stanford University - Main Campus - Lane Medical Library on 06/19/13. For personal use only.
Annu. Rev. Genom. Human Genet. 2005.6:331-354. Downloaded from www.annualreviews.org
email: pinkel@cc.ucsf.edu, albertson@cc.ucsf.edu
Key Words array CGH, clinical genetics, cancer genetics, genomic instability,
genome profiling
■ Abstract Altering DNA copy number is one of the many ways that gene expres-
sion and function may be modified. Some variations are found among normal individ-
uals (14, 35, 103), others occur in the course of normal processes in some species (33),
and still others participate in causing various disease states. For example, many defects
in human development are due to gains and losses of chromosomes and chromoso-
mal segments that occur prior to or shortly after fertilization, whereas DNA dosage
alterations that occur in somatic cells are frequent contributors to cancer. Detecting
these aberrations, and interpreting them within the context of broader knowledge, fa-
cilitates identification of critical genes and pathways involved in biological processes
and diseases, and provides clinically relevant information. Over the past several years
array comparative genomic hybridization (array CGH) has demonstrated its value for
analyzing DNA copy number variations. In this review we discuss the state of the art
of array CGH and its applications in medical genetics and cancer, emphasizing general
concepts rather than specific results.
INTRODUCTION TO ARRAY COMPARATIVE

GENOMIC HYBRIDIZATION
The development of comparative genomic hybridization (CGH) (18, 44) provided
the first efficient approach to scanning entire genomes for variations in DNA
copy number (Figure 1a). In a typical CGH measurement, total genomic DNA is
isolated from test and reference cell populations, differentially labeled, and hy-
bridized to a representation of the genome that allows the binding of sequences at
different genomic locations to be distinguished. More than two genomes can be
compared simultaneously if suitable labels are available. Hybridization of highly
repetitive sequences is typically suppressed by the inclusion of unlabled Cot-1
DNA in the reaction. Originally, metaphase chromosomes were used for the rep-
resentation of the genome and the location of copy number variations between test
and reference genomic DNA was mapped to the physical position on the chro-
mosomes. Now chromosomes have largely been replaced by DNA microarrays
containing elements that are mapped directly to the genome sequence (70, 71, 85).
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332 PINKEL ALBERTSON
The relative hybridization intensity of the test and reference signals at a given lo-
cation is then (ideally) proportional to the relative copy number of those sequences
in the test and reference genomes. If the reference genome is normal then increases
and decreases in signal intensity ratios directly indicate DNA copy number varia-
tion within the genome of the test cells. Data are typically normalized so that the
modal ratio for the genome is set to some standard value, typically 1.0 on a linear
scale or 0.0 on a logarithmic scale. Additional measurements such as fluorescent
in situ hybridization (FISH) or flow cytometry (62) can be used to determine the
actual copy number associated with a ratio level.
Array CGH has been implemented using a wide variety of techniques. The
initial approaches used arrays produced from large-insert genomic clones such as
bacterial artificial chromosomes (BACs) (70, 85). Producing sufficient BAC DNA
of adequate purity to make arrays is arduous, so several techniques to amplify
small amounts of starting material have been employed. These techniques include
ligation-mediated polymerase chain reaction (PCR) (84), degenerate primer PCR
using one (34) or several (24) sets of primers, and rolling circle amplification (80).
BAC arrays that provide complete genome tiling paths are now available (43, 47a,
50). Arrays made from less complex nucleic acids such as cDNAs (71), selected
PCR products (17, 59), and oligonucleotides (7, 10) are also used. Although most
CGH procedures employ hybridization with total genomic DNA, some use re-
duced complexity representations of the genome produced by PCR techniques.
Computational analysis of the genome sequence is used to design array elements
complementary to the sequences contained in the representation (55). Currently,
various single nucleotide polymorphism (SNP) genotyping platforms, some of
which use reduced complexity genomic representations, are being evaluated for
their ability to determine both DNA copy number and allelic content across the
genome (106, 107).
The different basic approaches to array CGH provide different levels of per-
formance, so some are more suitable for particular applications than others. The
factors that determine the performance requirements include the magnitudes of
the copy number changes, their genomic extents, the state and composition of the
specimen, how much material is available for analysis, and how the results of the
analysis will be used (Figure 1b). Many applications require reliable detection
of copy number changes of much less than 50%, a more stringent requirement
than for other microarray technologies. Note that technical details are extremely
important and different implementations of the “same” array CGH approach may
yield different levels of performance.
TECHNICAL CONSIDERATIONS
Hybridization Signals
The major technical challenge of array CGH is generating hybridization signals
that are sufficiently intense and specific so that copy number changes can be de-
tected. The signal intensity on an array element is affected by a number of factors
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including the base composition, the proportion of repetitive sequence content,

and the amount of DNA in the array element available for hybridization. Thus,
intensities may vary by a factor of 30 or more among array elements even if
there are no copy number changes. If the entire measurement process behaves
ideally (that is, the signals are linearly proportional to sequence abundance), then
the comparative hybridization strategy of Figure 1 provides ratios that are quan-
titatively proportional to copy number. Most importantly, production variability
among different arrays, such as the amount of DNA in array elements, is accu-
rately compensated. Ratio accuracy is maintained even if the intensities become
nonlinearly related to genomic abundance due to processes that affect the test
and reference genomes equally, such as saturation of array elements or reassoci-
ation of double-stranded nucleic acids during hybridization (69). The alternative
strategy—hybridization of a single genome to an array and comparison of the
result to a set of historical controls—places more stringent requirements on repro-
ducibility of array manufacture and hybridization conditions to avoid reduced data
quality.
The complexities of the genomic DNA and of the DNA in the array elements
significantly affect signal intensities and thus play a dominant role in determining
the genomic resolution of different array CGH technologies. For example, copy
number information from genomes such as bacteria and yeast (28, 100) is easer
to obtain than from mammalian genomes, which are 100 to 1000 times larger,
because the concentration of each portion of the genome in the hybridization is
correspondingly higher. Similarly, due to a number of complex kinetic factors, ar-
ray elements made from genomic BAC clones (complexity ∼100–200 kb) typically
provide more intense signals than elements employing shorter sequences such as
cDNAs, PCR products, and oligonucleotides. The higher signals from the more
complex array elements result in better measurement precision, allowing detection
of single-copy transition boundaries—even in specimens with a high proportion
of normal cells—and localization of copy number transitions to a fraction of the
length of the array element in some circumstances (3).
Arrays with low complexity elements can potentially provide better genomic
resolution than BAC arrays if their measurement precision is adequate for the ap-
plication. The advantages of using shorter sequences, including the opportunity
to design arrays directly from the genome sequence, the ability to use the same
arrays for expression and genomic analysis, and the possibility of higher genomic
resolution, drive efforts to improve array performance with low complexity ele-
ments. Single-copy changes on individual array elements have been detected on
sequences as short as several kilobases (59), and even several hundred kilobases
(17), but that is not currently possible with oligo arrays (4, 10, 77). Figure 2 il-
lustrates the relationship of measurement precision and genomic resolution for
analyzing a single-copy deletion boundary using arrays of elements made from
BACs, fosmids, and PCR products of several kilobases in length. As indicated
above, some measurement approaches reduce the complexity of the genomic DNA
in order to increase signal intensities and allow the use of low complexity array
elements (77, 106, 107). Published data from these procedures indicate the noise
levels are too high to allow detection of single-copy changes affecting single array
elements.
Genome Characteristics and Copy Number Measurement

The change in ratio produced by a copy number change is affected by several
intrinsic characteristics of the specimen DNA. Most important are the high-copy
repetitive sequences dispersed throughout mammalian genomes. These can hy-
bridize to array elements that contain copies of the repeats, such as those made
from genomic and cDNA clones, overwhelming the signal due to the unique se-
quences. Hybridization from these sequences must be blocked, typically by adding

unlabeled Cot-1 DNA to the hybridization, or the repetitive sequences need to be
removed from the genomic DNA or be absent from the array elements. Blocking
is not perfectly effective so signals are biased by the residual repetitive sequence
hybridization. In addition, there may be general nonspecific binding of test and
reference genomic DNA to the array elements.
A simple model for the effect of biases that equally affect the test and reference
signals, such as the bias produced by unsuppressed repetitive sequences, shows
that the ratio is linearly related to copy number, but the slope decreases as the
bias increases because the signal does not become zero even if all copies of locus
are absent (see Figure 3a) (62, 68, 69). Linear ratio increases over many orders
of magnitude have been demonstrated in some array CGH systems (70). Figure
3b shows data from a cell line that is reasonably homogeneous in its genomic
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constitution and has true copy number levels ranging from 0 to 3 as determined by
FISH. The measured ratios for this sample, and published data (98), demonstrate
that this simple model provides an accurate description of the behavior of some
←
Figure 3 Relationship of measured ratios to copy number change. (a) Calculated ra-
tios (linear representation on the left, logarithmic on the right) are shown as a function
of copy number using a simple model (68) that includes the signal from unsuppressed
repetitive sequences and nonspecific hybridization. The ratios are plotted relative to
the normalized copy number, which is set to 1.0 for the median copy number in the
genome. The heavy line shows the dependence when the signal is entirely due to se-
quences uniquely associated with the locus corresponding to the array element. The
five lighter lines show the dependence when test and reference signals on the array
element include a bias equal to 10%, 20%, 30%, 40%, or 50% of the signal that would
be present when the normalized copy number of the locus is 1. The round circles
indicate the ratios corresponding to true copy number of 0, 1, 2, and 3 found in the
profile in part b of the figure. [The model assumes that the bias, β, is proportional to
the total amount of genomic DNA used in the hybridization, but independent of the
copy number of a particular locus because it is generated by sequences distributed
throughout the genome. Because the unique sequence signal on an array element is
also proportional to the amount of genomic DNA, after normalization one can write
that the test signal is C + β, where C is the copy number of the locus normalized
to the median, or any other similar value, for the genome, and the reference signal
is 1 + β. Thus, ratio = (C + β) / (1 + β). Lines show behavior for β = 0, 0.11,
0.25, 0.43, 0.67, and 1.0]. (b) Ratio profile of a variant of cell line HCT-116 while
undergoing selection for resistance to methotrexate (83). Array comparative genomic
hybridization (CGH) was performed using the bacterial artificial chromosome (BAC)
arrays, and actual copy number levels for the parental HCT-116 cells were previously
determined using fluorescent in situ hybridization (FISH) (84). The ratios were directly
calculated from the background-corrected test and reference signal intensities for each
element. An overall normalization factor was applied to set the median Log2 ratio = 0.
No other computational adjustments were employed. The cell line contains a well es-
tablished homozygous deletion on chromosome 16p, Log2 ratio ∼−3.2 in this analysis,
as well as single-copy deletions, Log2 ratio ∼−0.8, and single-copy gains, Log2 ratio
∼0.5. Plotting these points on part a of the figure demonstrates that in this data set
the typical bias on the array elements was equal to approximately 10% of the diploid
signal level, and the response slopes for all array elements were very similar. Individual
clones with ratios much different from 0 indicate copy number polymorphisms, focal
aberrations, or noise. Close examination of the ratios indicates that some genomic
regions are heterogeneous in copy number in this population, presumably due to the
ongoing selection. In particular, the ratio on chromosome 5q, the site of the DHFR
gene, the target of methotrexate, is slightly higher than other regions of the HCT116
genome that are characteristically present at 3 copies. (Unpublished data courtesy of
A. Snijders.)
array CGH systems. If the magnitude of the biases differs significantly among
array elements, for example due to different repetitive sequence content, then the
elements will reproducibly follow different curves in Figure 3a. Such behavior may
lead to false indications of recurrent copy number structure within a region where
the aberrant copy number is actually constant, thus producing false indications of
the potential locations of critical genes.
The performance of an array system for measuring heterogeneous specimens,
for example tumor specimens containing normal cells, can be estimated by first
establishing its behavior with a well characterized homogeneous specimen to de-
termine the effective bias level (see Figure 3). The expected ratio changes in the
heterogeneous specimen can then be obtained using the measured response curve
in conjunction with values of the normalized copy number appropriate for the
aberrations in the specimens to be measured. For example, a single-copy loss in a
homogeneous (near) diploid cell population results in a normalized copy number
of 0.5, whereas it is 0.75 if that cell population is mixed with an equal number of
normal cells. Comparing the expected ratio changes with the noise level expected
for the specimen then allows determination of the ranges of copy number change
and specimen heterogeneity for which acceptable performance might be expected.
Finally, this simple model does not describe the behavior of the measurements if
the effective biases on array elements have contributions from autofluorescence,
differential nonspecific behavior due to the labels in the genomic DNA, high levels
of nonspecific binding to the array substrate, or if the measurement process has ar-
tifacts introduced by nonlinearities in the imaging systems or characteristics of the
image analysis software. These effects may lead to very complex and idiosyncratic
behavior among the array elements.
CGH measurements are also affected by low-copy reiterated sequences that
are common to all individuals. Low-copy reiterated sequences include members
of gene families and blocks of duplicated sequences (20, 21, 56). If a locus that
contains such a sequence is changed in copy number, the corresponding ratio
change may underestimate the magnitude of the aberration because the other loci
with copies of that sequence remain at normal copy number (105). Conversely, all
loci that contain a copy of the sequence may show a ratio change when one locus
is altered (54, 98).
Specimen Preparation
The quality of genomic DNA preparations has a substantial effect on the resulting
data. Although isolating genomic DNA from fresh and frozen specimens is routine
through use of numerous published protocols and commercial kits, there appears to
be an unknown class of contaminants that occasionally copurify with the DNA and
produce abnormally high “noise” in the ratios. This noise is typically not random
because relabeling a different aliquot of the same DNA reproduces exactly the same
noisy pattern. Repurifying or, better, reisolating the DNA may help significantly.
DNA quality issues are especially acute when analyzing formalin-fixed archival
tissue. Data obtained from such specimens can range from excellent, basically
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indistinguishable from fresh tissue, to unusable. Simple diagnostics such as the

length distribution of the isolated DNA have not been reliable predictors of per-
formance. One difficulty may be determining the amount of DNA that is actually
present in a specimen because contaminants from the tissue section or isolation
procedure may interfere with standard fluorimetry and absorbance measurements.
Some investigators use PCR to assess the quantity of “effective” DNA in a spec-
imen (16). Empirically, increasing the amount of DNA by a factor or two in the
labeling reactions often improves results (82).
The amount of specimen DNA is frequently a constraint on CGH measure-
ments. Typical array CGH procedures use 300 ng to 3 µg of specimen DNA in the
labeling reaction, equivalent to ∼50,000 to 500,000 mammalian cells. Most proto-
cols employ random primer labeling, which also amplifies the DNA, so that several
micrograms are used in the hybridization. The need to obtain analyses from small
specimens, or small regions of heterogeneous specimens, has motivated significant
efforts to develop whole-genome amplification procedures. The strand displacing
polymerase φ29 has been used when the genomic DNA is present in long frag-
ments, permitting analysis of nanogram quantities (36, 48). Several companies
offer kits for such amplifications. DNA from formalin-fixed specimens is typically
too short for this approach. A number of other procedures including degenerate
primer PCR (12, 16), two-stage random primer labeling reactions (16), balanced
PCR (96), ligation-mediated PCR (31, 52, 87), and ligation circularization of de-
graded DNA (97) have also been used for DNA from both fresh/frozen and fixed
specimens. The PCR generation of genomic representations used in some tech-
niques also amplifies the DNA, allowing analysis of tens to hundreds of nanograms
of input DNA (77, 106, 107). The judgment on how well any of these techniques
work depends on the requirements of the desired application (Figure 1b).
Data Analysis
A number of primary processing approaches have been applied to obtain ratio
profiles. Normalization in some cases involves only a simple overall factor to set
the median ratio to some standard value, whereas in others additional procedures
based on spatial and intensity dependence and historical data specific to each array
element may also be applied. Occasionally, genomes have so much copy number
variation (Figure 4, upper panel;) that the biological significance of the normal-
ization is uncertain because only a very small proportion of the genome is at the
normal ratio. Some platforms use data from a single hybridization, whereas others
combine data from two measurements with dye reversal. Using data adjustment
procedures without understanding the underlying processes responsible for the dis-
tortions, or a robust phenomenological validation that the procedures are stable and
give reasonable results in control specimens, runs the risk of introducing systematic
errors.
Although the major aberrations in a genome are frequently evident by inspec-
tion, many approaches to improve interpretation in the face of measurement noise
have been developed. The simplest is to apply thresholds. If the ratio profile has
only a few well spaced ratio levels, thresholds can be chosen by examining the
distribution of all measured ratios (34). However, many tumors, owing to their
nondiploid genomes and/or heterogeneity, have closely spaced ratio levels that
partially overlap due to measurement noise, so this approach is not capable of dis-
tinguishing them. Smoothing by averaging the ratios on neighboring array elements
improves the behavior of thresholding but blurs the locations of boundaries and
reduces the amplitude of aberrations involving fewer elements than the smoothing
window.
More sophisticated analytical approaches employ the fact that the copy number
changes involve chromosome segments, so ratios at contiguous sets of loci should
be identical, except for occasional abrupt steps to a new level. These methods
statistically assess the status of each array element in the context of its neigh-
bors. Among the approaches that have been used are Hidden Markov Models (27);
change point analysis (66), adaptive weights smoothing (38), Baysean maximum a
posteriori probabilities (13), and ratio clustering (99), and many more are being de-
veloped. Several software packages are available at http://www.bioconductor.org/.
Statistical approaches limited to examination of ratio profiles cannot evaluate the
reliability of an aberrant ratio that affects only a single array element. The underly-
ing image data needs to be examined to determine its quality, and the interpretation
needs be accomplished in light of experience. Single-copy aberrations that affect
only one array element can be detected with high sensitivity and specificity with
some BAC technologies and may be highly informative (82).
APPLICATIONS
Variation in Normal Genomes
Differences in gene structure and variability in gene families produce DNA dosage
polymorphisms in the human genome (14, 35). A comprehensive understanding
of these normal variations is of intrinsic biological interest and is essential for
proper interpretation of array CGH data and its relation to phenotype. Array CGH
measurements using BAC arrays immediately revealed copy number polymor-
phisms (2). Figure 5 illustrates ratio variation in a publicly available data set (84)
for several clones that are contained in well-known polymorphisms. Each of these
clones shows a different magnitude and frequency of variation across the cell lines.
An analysis by Iafrate et al. (41) of 39 normal individuals with a different set of
∼2500 BACs found that about 10% of clones showed ratios consistent with a copy
number change in at least one individual, and on average each person had 12.4
variant ratios. About 100 clones showed variant ratios in more than one person,
with the most polymorphic loci showing variability in about half of the people.
Polymorphisms have also been detected using the representational oligonucleotide
microarray analysis (ROMA) approach to CGH (77). ROMA samples the genome
differently than BAC arrays because it employs reduced complexity genomic
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representations in the hybridization. Analysis of 20 individuals found 221 pu-

tative copy number polymorphisms at 76 loci. Great ape genomes have sufficient
sequence homology to human to be analyzed on human BAC arrays (53). More
than 60 putative sites of large-scale sequence variation among human, chimpanzee,
bonobo, gorilla, and orangutan were found. These were biased toward sites of seg-
mental duplication in the human genome, indicating the evolutionary importance
of such events. As described below, segmental duplications are also mechanisti-
cally important in formation of health-related genetic abnormalities in humans.
Although CGH has established the prevalence of copy number polymorphisms
in the human genome, the picture of this normal variation is incomplete. In results
reported to date, measurement noise has restricted detection to polymorphisms that
involve genomic segments of many kilobases or larger, genome coverage has been
far from comprehensive, and the population has not been adequately sampled. The
published studies also raise questions concerning their mutual consistency. For
example, Iafrate et al. (41) report three times as many distinct polymorphic loci on
their BAC arrays than do Sebat et al. (77), even though Sebat et al. use arrays with
30 times as many elements. Part of this difference may be due to the analytical
procedures and decision criteria employed in the two studies, and to the differences
in their technical limitations. The further elucidation of dosage polymorphisms
will remain an experimental rather than computational endeavor until high-quality
sequence is available on a large number of individuals. Understanding the copy
number polymorphisms that are detectable by a particular array CGH technique
is important so that normal variations are not falsely associated with disease,
and, conversely, to determine if some so-called normal variation may underlie
phenotypic characteristics such as disease susceptibility (89).
Mouse genomes also have a high frequency of copy number polymorphisms.
Comparative hybridization of 14 M. musculus strains using arrays of ∼19,000
mouse BACs selected to produce a tiling path of the sequenced portion of the
mouse genome found ∼350 clones that detected polymorphisms (50). Of these,
216 consistently showed loss relative to C57BL6, and 130 consistently showed
gains, with very few showing both gains and losses among the strains. Thus, about
2% of the clones were classified as polymorphic among the strains. Snijders et al.
(81) found a much higher frequency of apparent polymorphisms in the analysis of
multiple individual mice from seven M. musculus and two M. spretus strains using
arrays of ∼2000 clones, ∼1300 of which were nonredundant and nonoverlapping.
About 6% of these clones showed |log2 ratio| > 0.4 in one of six M. musculus strains
when compared to FVB/N. Clustering of the mouse strains based on either set of
polymorphisms was consistent with the known evolutionary relationships among
the strains. Although these two papers clearly demonstrate the presence of a large
number of copy number polymorphisms among the inbred mouse strains, there is
no obvious concordance among the sets of clones. Among the factors that could
contribute to the differences in the two studies is the method of clone selection.
The tiling arrays did not include portions of the genome that were not in the
physical map, and the smaller arrays were assembled prior to the existence of the
physical map and comprised BACs with mapped STS markers. Thus, the arrays of
Snijders et al. (81) might include regions of complex, and perhaps polymorphic,
genome sequences that are difficult to assemble. There may also be significant
differences in the criteria used to classify clones as polymorphic, but this remains
to be determined.
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The presence of small-scale, highly distributed polymorphisms among mouse

strains and species has also been detected using BAC arrays (81). The measure-
ments are based on the fact that sequence homology as well as abundance affects
signal intensities. The sequence divergence produces two effects. First, comparing
normal genomes of various M. musculus and M. spretus strains to an array made
from a M. musculus BAC library found that the ratio variation across the genome
increased with increasing evolutionary distance between the mice, presumably
due to the expanding number and uneven distribution of polymorphisms across
the genome. Similar effects may be detectable among widely divergent human
populations. Second, comparing DNA from (NIH × SPRET/Glasgow) F1 × NIH

backcross mice using an NIH reference found slightly lower ratios for the por-
tions of the genome that were heterozygous NIH/SPRET than for those that were
homozygous NIH. These measurements provide efficient, high-resolution map-
ping of the chromosomal constitution of the backcross animals without the need
for knowledge of specific markers. This technique will facilitate investigations
of genotype/phenotype relationships using interspecific backcrosses (for example,
identifying cancer susceptibility and resistance alleles). Detecting loss of heterozy-
gosity in F1 animals should also be possible. The minimum amount of sequence
divergence required for this procedure has not been established.
←
Figure 5 Examples of human DNA copy number polymorphisms. Each panel shows
the ratio values for array elements [bacterial artificial chromosome (BAC) clones] from
polymorphic loci in 15 different cell strains obtained from the Coriell Cell Repository.
The gray bands indicate the range of ratio variability for nonpolymorphic loci. Data
are from Snijders et al. (84) and are publicly available. The names of the BAC clones
used for the array elements are indicated in each panel. (a) The highly polymorphic
Lipoprotein(a) locus (14) on chromosome 6q shows a different ratio in nearly every
individual. This polymorphism is due to variation in the numbers of a 5-kb repeat
sequence in the gene. (b) Two partially overlapping BAC clones in the Defensin gene
cluster (35) on 8p show similar copy number variation in most individuals. However,
in several individuals there are clear differences in ratios of the clones, indicating
that a polymorphic structure occasionally affects the nonoverlapping portions of the
clones differently. The preponderance of negative ratios in these individuals presumably
reflects status of this polymorphism in the DNA used for the reference. For example,
if a person similar to GM04435 had been the reference, all others would have shown
positive ratios. (c) A polymorphism on 8q behaves as if there are two alleles in the
population (103). Most individuals have the genomic region present on both of their
chromosomes. Two individuals have a deletion that includes most or all of the BAC
on one of their copies of chromosome 8, based on the observed log2 ratio of ∼−1. One
individual is homozygous for the deletion, based on the observed very low ratio. (See
Figure 3 for dependence of ratio on copy number.)
Dosage Abnormalities in Developmental Genetics

CLINICAL STUDIES Many individuals with developmental abnormalities have
chromosomal aberrations that result in dosage changes for parts of the genome. For
example, trisomies of chromosomes 13, 18, and 21 and copy number abnormalities
involving the sex chromosomes are the most frequent constitutional abnormali-
ties affecting newborns. These are the only whole-chromosome aneuploidies that
are compatible with live birth. Many others are detected in spontaneous miscar-
riages. Additional aberrations such as the presence of supernumerary chromo-
somes, segmental duplications and deletions, and structural rearrangements are

also frequently found. Currently, cytogenetic analysis of metaphase chromosomes
and FISH are the most common methods for screening for abnormalities. These
methods are very powerful, but have significant limitations. Standard cytogenetics
provides a whole-genome scan capable of detecting gains and losses of chromo-
somes or chromosomal regions and structural rearrangements within and between
chromosomes. However, the identification of aberrations requires interpretation of
chromosome banding patterns, which may be ambiguous, and limits resolution to
several megabases at best. The addition of multicolor chromosome staining using
multicolor fluorescence in situ hybridization (M-FISH) or spectral karyotyping
(SKY) can resolve some ambiguities and detect smaller aberrations, but these ap-
proaches are still limited in resolution (51). FISH with probes for specific loci,
for example subtelomeric sequences, has very high resolution but the labor that is
required limits the number of loci that can be addressed (15, 60). These limitations
were among the factors that motivated development of array CGH.
Array CGH has proven very effective for highly parallel screening of genomes
for clinically significant dosage abnormalities. Detection of the most prevalent
abnormalities, the whole-chromosome aneuploidies described above, is straight-
forward. More complex analyses of specific chromosomal loci have also been
demonstrated with high reliability. The established clinical significance of telom-
eric abnormalities that are undetectable by standard cytogenetics (47), and the
need for highly parallel analysis of these many loci, has resulted in construction of
arrays containing unique subtelomeric sequences for essentially all chromosome
ends. The array results have proven very specific and sensitive. Use of multiple
closely spaced array elements in the telomeric regions to detect events missed by
the standard FISH tests has provided a more detailed determination of the nature
of the aberrations and their potential phenotypic effect (92, 102). Although many
miscarriages have cytogenetically detectable chromosomal aberrations, many do
not. Array CGH using an array that provided targeted rather than comprehensive
coverage of the genome detected dosage aberrations in an additional 10% of cases,
indicating a probable reason for the spontaneous termination of the pregnancy (75).
Many investigators have analyzed genomic DNA from developmentally abnor-
mal individuals to determine if they have cytogenetically cryptic chromosomal
abnormalities. The challenging aspect of these studies is that the aberrations are
likely to be subtle, so measurement noise and polymorphisms may provide false
positive indications of causal abnormalities. One study reported detection of copy
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number changes in 10% to 25% of cases (79, 93), leading to the identification of the
critical gene involved in coloboma, heart defects, atresia choanae, retarded growth
and development, genital hypoplasia, ear anomalies and deafness (CHARGE) syn-
drome (94). Even when aberrations are cytogenetically detectable, array CGH pro-
vides a significantly enhanced ability to map the events in multiple cases, thereby
facilitating identification of the critical gene (86). Numerous studies are underway
to use array CGH for analysis groups of phenotypically abnormal patients with
normal karyotypes. Many of these studies remain unpublished, pending confirma-
tion from family analyses that the aberrations are likely causal or due to the lack of
candidate aberrations. The success in finding aberrations in various series of pa-

tients has varied widely, presumably due to whether the phenotypes of the patients
were actually due to unbalanced chromosomal aberrations, the degree of genomic
coverage provided by the array, and the performance of the array platform. As the
effective resolution of array CGH techniques increases, one expects an increasing
rate of discovery of medically important dosage aberrations. However, as indicated
above, interpreting the primary data will become more complex due to the need to
better understand the “normal” polymorphisms in the genome.
Although most studies to date have involved postnatal analysis, prenatal ap-
plications of array CGH are very attractive. Because the analysis requires only
genomic DNA, success will depend on whether sufficient high-quality DNA can
be obtained, and whether that DNA reflect the status of the fetus. Ki et al. (45)
used DNA from uncultured amniocytes to detect a dosage abnormality due to
a ring chromosome, and Larrabee et al. (49) demonstrated the ability to use
cell-free DNA from the amniotic fluid for effective analysis. Reproductive tech-
nologies may also benefit from array CGH analysis. If adequate DNA amplifica-
tion and measurement techniques can be developed, reliable single-cell analysis
of preimplantation embryos will be possible. Hu et al. (37) took a step in this
direction using arrays of elements that contain DNA representative of entire
chromosomes. These high-complexity elements give sufficient signal intensity to
demonstrate the potential of the approach for detecting whole-chromosome ane-
uploidies, but the reliability needs to be substantially increased prior to clinical
application.
One critical question raised by conventional cytogenetic analysis is whether or
not an apparently balanced structural rearrangement is accompanied by cryptic
dosage imbalances or breakpoints that could have phenotypic effects. Array CGH
with ∼1-Mb resolution arrays detected a high frequency of small deletions and
duplications in these cases (30, 46, 88). Higher resolution analysis would probably
yield even more abnormalities. Although array CGH with whole genomic DNA
cannot provide information on truly balanced structural rearrangements, analysis
of isolated chromosomes, obtained, for example, by flow sorting, can be employed
efficiently to determine the composition of abnormal chromosomes and to map the
breakpoints (25, 29, 30, 91). These studies frequently found unexpected complexity
in the breakpoints.
Proper implementation and interpretation of array CGH data in the clinic
presents a range of important issues that need to be addressed. From the standpoint
of discovery, one would like to perform the highest resolution studies that are pos-
sible in order to gather the information required to assess phenotypic effects of
particular aberrations. However, one might find apparent abnormalities whose con-
sequences are unknown, an especially acute problem for prenatal analysis. One
approach is to use arrays that only contain elements designed to detect aberrations
whose interpretation is well established (5, 75). However, even when the arrays
are targeted, the CGH analysis may not provide sufficient information to inter-
pret the potential phenotypic consequences of an aberration. Although FISH is
frequently discussed as a method to simply validate array results, its more funda-
mental role will likely be in performing additional studies suggested by the array
results in order to obtain additional essential diagnostic and counseling informa-
tion (102). Establishing the proper implementation of array CGH for different
clinical applications will require considerable thought because excessive caution
will inappropriately restrict the benefits that can be obtained, whereas proceeding
without sufficient consideration will lead to significant errors.
MECHANISMS OF ABERRATION FORMATION Some recurrent constitutional abnor-

malities are caused by rearrangements mediated by sequence duplications (i.e.,
“duplicons”) that are present at many locations in the genome (19, 21, 56). Chro-
mosome 15 has a high frequency of clinically significant dosage abnormalities and
a high frequency of duplicons. High-resolution array CGH analysis of the Prader-
Willi/Angleman region at 15q11–13 examined the details of duplicon-mediated
rearrangements in patients (54, 98). Analysis of chromosome 17p, the site of four
disorders caused by deletions or gains of sequence, has also been reported (78).
The arrays allowed proper quantification of the losses, duplications, and triplifi-
cations of sequences and detection of unusual asymmetric rearrangements. Array
elements that contained duplicated sequences behaved as expected, reporting the
overall genome content of the sequences rather than the local sequence copy num-
ber. Apparent polymorphisms within the duplicated regions may also have been
detected. Array CGH is limited in its analysis of this genomic region because
Prader-Willi syndrome results from the lack of expression of paternally inherited
genes. Thus, events that do not affect copy number, such as maternal uniparental
disomy, can cause the disease. Perhaps techniques now under development that
combine SNP and copy number analyses will allow more comprehensive detection
of significant chromosomal aberrations in 15q (106, 107).
GENOTYPE/PHENOTYPE RELATIONSHIPS Determining genotype/phenotype rela-

tionships in contiguous gene syndromes is a critical step in localizing the genes
involved in different aspects of the phenotype and in understanding the functional
basis of the conditions. Array CGH has provided the ability to map aberrations
with high resolution and is particularly advantageous when large numbers of loci
need to be analyzed. For example, a tiling path array of BAC clones on chro-
mosome 4p was used to map deletions in Wolf-Hirschron syndrome (90). The
facial characteristics of this phenotype were attributed to a single gene, but other
ARRAY CGH 345
characteristics were due to the action of multiple genes. Zhang et al. (105) used a
high-resolution array of clones on chromosome 5p to analyze deletions in Cri du
Chat syndrome. They refined the localization of the regions of the chromosome,
which, when deleted, contributed to various aspects of the phenotype, including
the typical cry, facial features, and speech delay. In addition, they demonstrated
the interaction of various regions of 5p in producing the mental retardation phe-
notype, and, using whole-genome scanning, found that the most severely retarded
individuals frequently had dosage abnormalities in addition to 5p deletions. Array
CGH has also been used to investigate genomic alterations associated with neurofi-
bromatosis type 2 (NF2). This autosomal dominant disorder displays pronounced

heterogeneity in formation of benign and malignant tumors. Using targeted arrays
for regions of chromosome 22, it was found that the type of mutation in the NF2
gene and the patient phenotype were not correlated (8), and deletions in tumors
presented a complex picture, sometimes not including the NF2 gene (58).
Genomic Analysis of Cancer

Tumors develop through the combined processes of genetic instability and selec-
tion, resulting in clonal expansion of cells that have accumulated the most favorable
(for the tumor) set of genetic aberrations. Many types of instability may occur,
resulting in production of point mutations, chromosomal rearrangements, DNA
dosage abnormalities, altered microsatellite sequences, and epigenetic changes
such as methylation, etc. These abnormalities act alone or in combination to al-
ter the functions and/or expression levels of cellular components. Fully developed
tumors contain the genetic history of their development, but this history may be dif-
ficult to decipher. Some aberrations that are significant early in tumor development
may be lost or obscured by subsequent events or may no longer be functionally
relevant. Others may be neutral or even somewhat detrimental to the tumor but
are found because they were also present in a cell that developed a sufficiently
protumorigenic aberration, or because they are obligate products of the event that
produced a critical aberration. Array CGH provides a powerful entry point for
studies of cancer, owing to its ability to analyze DNA from a wide variety of spec-
imens, including those that are not amenable to other forms of global analysis.
The leads that are generated frequently motivate follow-up studies that employ
the complete range of biological approaches, including expression analysis, im-
munohistochemistry, FISH, DNA sequencing, tissue microarrays, and functional
studies in tissue culture and animal models.
COPY NUMBER PHENOTYPES Tumor genomes reveal a wide variety of copy num-
ber “phenotypes” indicating different types of genetic instability. For example,
colon tumors have long been known to have different levels and types of genomic
aberrations (63), which were eventually attributed to differences in mismatch re-
pair competence (6, 22). Analysis of mismatch repair-proficient and -deficient cell
lines found that the exact nature of the repair deficiency also had significant effects
on the characteristics of the copy number changes (83). Figure 4 shows the DNA
copy number profiles of one breast cancer cell line and two breast tumors (1).
Clearly, different defects in the mechanisms that maintain genomic stability oc-
curred. Tumors in mouse model systems frequently do not contain large numbers
of informative genome copy number variations unless they were engineered to
have specific genetic defects such as impaired telomeres (65). The wide range of
genomic phenotypes in cancer means that for some sets of specimens array CGH
will provide significant information on the locations of important cancer genes,
whereas in others it will be uninformative. Note that copy number profiles of cell
populations reveal past genomic instability that lead to the clonal expansion of a
cell population with a relatively stable genome, at least stable within its selective
environment. Some tumors appear very stable in vivo, with the primary tumors
and recurrences having nearly identical copy number profiles even though there
are many years between them (1, 95). Ongoing genomic instability results in het-
erogeneity that is not detectable by CGH and is best assessed by techniques that
examine individual cells (11).
PROGNOSTIC UTILITY Knowledge of copy number aberrations can have immedi-

ate clinical use in diagnosis, and in some cases provide useful prognostic informa-
tion. Microarrays designed for profiling chronic lymphocytic leukemia have been
produced for use in clinical trials to facilitate determinations of the relationship
of therapeutic options with genomic aberrations (76). Association of DNA copy
number aberrations with prognosis has been found for a variety of tumor types,
including prostate cancer (67), breast cancer (9), gastric cancer (101), and lym-
phomas (61, 74). Many more studies are in press or nearing completion. As with
other types of statistical studies, these results require validation on independent
patient sets to control for the possibility of unanticipated systematic factors in the
initial patient groups (26, 42).
CANCER GENE IDENTIFICATION The identification of important genes within re-

gions of copy number change is a complex task. If narrow regions of highly ele-
vated copy number or total deletion that contain previously known cancer genes
or genes with suggestive function are found, high-probability candidates may be
immediately evident. Even if such aberrations are rare, they may suggest additional
measurements that develop support for the frequent involvement of a particular
candidate gene or pathway (82). However, in many cases, even minimally defined
aberrant regions resulting from combining data from many specimens may con-
tain several attractive candidates, or none, or the copy number aberrations may be
complex making it difficult to determine how many different loci may be under
selection. If a gain is greater than a single copy it is possible that more than one
evolutionary step was involved in its formation. This sometimes results in a profile
that resembles a peak with sloped sides, suggesting, but not proving, that the criti-
cal gene(s) are located in the vicinity of the top (3, 83). Thus, it is useful to interpret
the amplitude of copy number changes in addition to just noting their locations.
ARRAY CGH 347
Measurement of gene expression at the RNA or protein levels is critical for

candidate evaluation. One expects that if a gene is a target of selection within a
region of copy number increase it should be overexpressed in tumors in which
it is found at elevated copy number. Unfortunately, overexpression does not dis-
tinguish it from other genes in the aberrant region that may not contribute to the
tumor development because expression of 40% to 60% of all the genes may be
elevated (32, 40, 72). However, genes can be overexpressed for reasons other than
dosage increase, and therefore may be involved in tumor development even if at
“normal” copy number. Only rarely, the classic example being human ERBB2, are
expression changes at the RNA and protein levels essentially perfectly coupled to
dosage (73). Thus, finding that a gene is always overexpressed when at increased
copy number, and is sometimes overexpressed when not present at increased copy
number, supports its functional role in cancer. Genes that drive copy number gains
may also be altered by mutation (57), so sequencing candidates in tumors with and
without increases may provide critical information. Similarly, particular alleles of
a gene may contribute to tumorigenesis, so finding preferential gain of one variant
may indicate its functional involvement (23).
Evaluating genes in regions of copy number loss is also complex. In some cases
the decrease in expression due to the decrease in copy number is sufficient for the
gene to be significant to the tumor. However, in the classic case of tumor suppressor
genes function is totally abrogated by deletion of all copies of a gene, deletion of one
copy and mutation or epigenetic alteration of the other (104), or alteration of one
copy and replacement of the other by a duplicate of the altered copy, etc. The latter
type of aberration results in loss of heterozygosity but no copy number change,
and is not detectable by array CGH. The developing SNP profiling technologies
may be able to provide additional information concerning these events, perhaps
eventually providing information on heterozygosity and dosage for some types
of specimens (106, 107). Candidate genes within recurrent regions of loss can be
assessed for expression changes and examined to determine if the remaining copies
are mutated or methylated (104), etc. One general approach that has proven useful
to broadly screen for mutated genes in cells in culture employs nonsense-mediated
decay (NMD). If a mutation produces a premature stop codon the transcripts are
rapidly degraded (64). Global comparison of expression levels before and after
inactivating NMD identifies genes whose transcript levels have increased. Those
that are contained within deletions are attractive candidate tumor suppressors (39).
CONCLUSION
Array CGH is one of a growing number of top-down approaches that can provide
comprehensive information about aspects of biological status or function. In the
near term these techniques can provide correlational information that is useful
for clinical applications in oncology and medical genetics, and basic information
on fundamental characteristics of genome structure. The clinical applications in
medical genetics are particularly compelling and will be extensive in the immediate
future. Considerable care must be exercised to be sure that false positive indica-
tions of abnormality are kept to acceptable levels and that the aberrations that are
detected are interpreted with appropriate rigor. For the longer term, the combina-
tion of measurements of DNA copy number, RNA and protein expression levels,
mass RNAi screens in model systems, etc. will lead to substantial advances in fun-
damental understanding of biological processes. However, traditional bottom-up
studies of any one small component of a functional pathway always reveal details
that are not addressed by the global approaches. Conversely, focused studies may
be misinterpreted due to lack of global information. Thus, improving the ability

to integrate bottom-up and top-down information is essential. Looking back at
past accomplishments, and ahead to the increasingly powerful tools that continue
to become available, may lead to overoptimism about the ease of taking the next
steps. Profiling technologies can fill databases at prodigious rates. But they provide
little value unless the data are of sufficient quality and are rigorously evaluated and
interpreted within the richest possible context. In this review we tried to address
some of these issues as they pertain to array CGH.
AUTHORS’ NOTE
Portions of this manuscript are substantially similar to portions of a Perspective
that appeared in Nature Genetics volume 37, May 2005.
The Annual Review of Genomics and Human Genetics is online at

http://genom.annualreviews.org
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HI-RES-GG06-15-Pinkel.qxd
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Figure 1 (a) Array comparative genomic hybridization (CGH). Genomic DNA from two
cell populations is differentially labeled and hybridized to a microarray. The fluorescent
signal intensity ratios measured at each array spot are normalized so that the median
log2ratio 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 of a factor of two in copy number. Fluorescent in situ hybridization (FISH) with
a red-labeled probe for the deleted region and a green-labeled control probe (genome loca-
tions indicated by the red and green arrows on the ratio profile) shows that the cells con-
tain two copies of the green probe and only one for the red, consistent with the array CGH
analysis (84). (b) The difficulty of array CGH analysis varies significantly among different
applications. It is much easier to detect the large increase in copy number associated with
amplification of a genomic region than it is to detect single-copy gains or losses.
Aberrations affecting an extended genomic region including multiple array elements are
easier to detect than focal events. Measurements on cell lines are the least difficult because
isolation of high-quality DNA is straightforward and the genomes are relatively homoge-
neous. Fresh or frozen tumor tissue presents additional challenges due to possible tissue-
specific factors, and the potential for genomic heterogeneity in a tumor and/or inclusion of
normal cells. Measurements on formalin-fixed paraffin-embedded tissue present the great-
est challenges. Research studies aimed at profiling a group of tumor specimens that have a
large number of highly recurrent aberrations can be informative even if a considerable
number of errors are made in each tumor. In contrast, detection of aberrations that are both
rare and small, and of course clinical applications, present challenging specificity and sen-
sitivity requirements.

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ARRAY CGH
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Figure 2 Array comparative genomic hybridization (CGH) analysis of a deletion

boundary using arrays with elements of different complexity. Data from a bacterial
artificial chromosome (BAC) array (element complexity 150200 kb, black bars)
indicate the decrease in ratio due to the deletion. Data from a fosmid array (element
complexity 30–40 kb, green bars) provide more precise indication of the deletion
boundary, but the variation in ratios among the different array elements is increased.
Finally, data from an array of genomic polymerase chain reaction (PCR) products
(element complexity 1.5–4 kb, red bars) provide even higher resolution information
on the boundary, but with an even larger ratio variation. Noise from low complexity
array elements decreases in importance as the magnitude of the copy number change
increases, so if boundaries of amplified regions are abrupt they can be determined
even if the measurements are very noisy. Note that the data indicate that one of the
BAC clones is partially contained in the deletion, and that this might cause the slight-
ly reduced ratio on this clone. Thus, a tiling path of BAC clones could potentially
map the position of the copy number transition to a fraction of the length of a clone
(3). (Unpublished data courtesy of R. Redon.)
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Figure 4 Copy number profiles of breast cancer cell lines and primary tumors show
characteristic differences. The upper panel shows the BRCA1 mutant cell line HCC
1937 (1). Portions of almost every chromosome are present at different copy
numbers. The genome is so variable that the biological significance of the ratio nor-
malization is not clear. The middle and lower panels show profiles from two ductal
invasive breast tumors. These have large regions of the genome at the same copy
number, which provides a reference level from which to measure gains and losses.
The tumor in the middle panel contains only a few single-copy changes, whereas the
one in the lower panel has one region of high-level amplification in addition to lower
magnitude gains and losses (84).

P1: KUV
July 27, 2005 13:16 Annual Reviews AR252-FM
Annual Review of Genomics and Human Genetics

Volume 6, 2005
CONTENTS
A PERSONAL SIXTY-YEAR TOUR OF GENETICS AND MEDICINE,

Alfred G. Knudson 1
COMPLEX GENETICS OF GLAUCOMA SUSCEPTIBILITY, Richard T. Libby,
Douglas B. Gould, Michael G. Anderson, and Simon W.M. John 15
NOONAN SYNDROME AND RELATED DISORDERS: GENETICS AND
PATHOGENESIS, Marco Tartaglia and Bruce D. Gelb 45
SILENCING OF THE MAMMALIAN X CHROMOSOME, Jennifer C. Chow,
Ziny Yen, Sonia M. Ziesche, and Carolyn J. Brown 69
THE GENETICS OF PSORIASIS AND AUTOIMMUNITY, Anne M. Bowcock 93
EVOLUTION OF THE ATP-BINDING CASSETTE (ABC) TRANSPORTER
SUPERFAMILY IN VERTEBRATES, Michael Dean and Tarmo Annilo 123
TRADE-OFFS IN DETECTING EVOLUTIONARILY CONSTRAINED SEQUENCE
BY COMPARATIVE GENOMICS, Eric A. Stone, Gregory M. Cooper,
and Arend Sidow 143
MITOCHONDRIAL DNA AND HUMAN EVOLUTION, Brigitte Pakendorf
and Mark Stoneking 165
THE GENETIC BASIS FOR CARDIAC REMODELING, Ferhaan Ahmad,
J.G. Seidman, and Christine E. Seidman 185
HUMAN TASTE GENETICS, Dennis Drayna 217
MODIFIER GENETICS: CYSTIC FIBROSIS, Garry R. Cutting 237
ADVANCES IN CHEMICAL GENETICS, Inese Smukste
and Brent R. Stockwell 261
THE PATTERNS OF NATURAL VARIATION IN HUMAN GENES,
Dana C. Crawford, Dayna T. Akey, and Deborah A. Nickerson 287
A SCIENCE OF THE INDIVIDUAL: IMPLICATIONS FOR A MEDICAL SCHOOL
CURRICULUM, Barton Childs, Charles Wiener, and David Valle 313
COMPARATIVE GENOMIC HYBRIDIZATION, Daniel Pinkel
and Donna G. Albertson 331
SULFATASES AND HUMAN DISEASE, Graciana Diez-Roux
and Andrea Ballabio 355
v
P1: KUV
July 27, 2005 13:16 Annual Reviews AR252-FM
vi CONTENTS
DISEASE GENE DISCOVERY THROUGH INTEGRATIVE GENOMICS, Cosmas

Giallourakis, Charlotte Henson, Michael Reich, Xiaohui Xie,
and Vamsi K. Mootha 381
BIG CAT GENOMICS, Stephen J. O’Brien and Warren E. Johnson 407
INDEXES
Subject Index 431
Cumulative Index of Contributing Authors, Volumes 1–6 453
Cumulative Index of Chapter Titles, Volumes 1–6 456

ERRATA
An online log of corrections to Annual Review of Genomics
and Human Genetics chapters may be found
at http://genom.annualreviews.org/

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Annu. Rev. Genomics Hum. Genet. 2005. 6:331–54

COMPARATIVE GENOMIC HYBRIDIZATION

email: pinkel@cc.ucsf.edu, albertson@cc.ucsf.edu

INTRODUCTION TO ARRAY COMPARATIVE

332 PINKEL ALBERTSON

ARRAY CGH 333

including the base composition, the proportion of repetitive sequence content,

334 PINKEL ALBERTSON

Genome Characteristics and Copy Number Measurement

quences. Hybridization from these sequences must be blocked, typically by adding

ARRAY CGH 335

336 PINKEL ALBERTSON

ARRAY CGH 337

indistinguishable from fresh tissue, to unusable. Simple diagnostics such as the

338 PINKEL ALBERTSON

ARRAY CGH 339

representations in the hybridization. Analysis of 20 individuals found 221 pu-

340 PINKEL ALBERTSON

ARRAY CGH 341

The presence of small-scale, highly distributed polymorphisms among mouse

populations. Second, comparing DNA from (NIH × SPRET/Glasgow) F1 × NIH

342 PINKEL ALBERTSON

Dosage Abnormalities in Developmental Genetics

somes, segmental duplications and deletions, and structural rearrangements are

ARRAY CGH 343

candidate aberrations. The success in finding aberrations in various series of pa-

344 PINKEL ALBERTSON

MECHANISMS OF ABERRATION FORMATION Some recurrent constitutional abnor-

GENOTYPE/PHENOTYPE RELATIONSHIPS Determining genotype/phenotype rela-

ARRAY CGH 345

bromatosis type 2 (NF2). This autosomal dominant disorder displays pronounced

Genomic Analysis of Cancer

346 PINKEL ALBERTSON

PROGNOSTIC UTILITY Knowledge of copy number aberrations can have immedi-

CANCER GENE IDENTIFICATION The identification of important genes within re-

ARRAY CGH 347

Measurement of gene expression at the RNA or protein levels is critical for

348 PINKEL ALBERTSON

be misinterpreted due to lack of global information. Thus, improving the ability

The Annual Review of Genomics and Human Genetics is online at

ARRAY CGH 349

8. Bruder CE, Hirvela C, Tapia-Paez I, formalin-fixed, paraffin-embedded breast

of array-CGH and tissue microarrays. J. of complete and partial chromosome gains

350 PINKEL ALBERTSON

ARRAY CGH 351

unbalanced translocation: characteriza- Esposito D, et al. 2000. Detecting gene

352 PINKEL ALBERTSON

46:143–56 copy number alteration in the transcrip-

ARRAY CGH 353

genomic hybridisation using a proximal at Xq27 containing SOX3. J. Med. Genet.

retardation and dysmorphic features. J. a cryptic interstitial 7q31.3 deletion in a

354 PINKEL ALBERTSON

chromodomain gene family cause nomas correlate with clinicopathological

See legend on next page

C-2 PINKEL ■ ALBERTSON

See legend on next page

C-4 PINKEL ■ ALBERTSON

Figure 2 Array comparative genomic hybridization (CGH) analysis of a deletion

See legend on next page

C-6 PINKEL ■ ALBERTSON