"No cookbook approach"
DNA Samples (PDF document 1,431 K)
In the laboratory sweepstakes, microarray-based comparative genomic hybridization
(array CGH) has the appearance of a winner. With the speed and power of
microarray technology and the focus and resolution of CGH, this newly
foaled technique has an excellent pedigree. Its ability to track down
constitutional chromosomal abnormalities puts it at the head of the pack.
Although array CGH has made only a few appearances in the clinical laboratory,
this new entry has already generated tremendous enthusiasm. Whether it
will earn a major financial prize, however, is a question that will be
settled outside the laboratory performance arena, in deliberations over
molecular devices at the FDA, issuance of new CPT codes, and determination
of Medicare reimbursement. There may also be disputes over just which
syndicate owns this high-performing thoroughbred. All in all, at the present
time no one can fix the odds on its success.
Array CGH is "totally revolutionizing chromosome diagnosis,"
says Arthur Beaudet, MD, chair of the Department of Molecular and Human
Genetics at Baylor College of Medicine, one of only two places in the
U.S. where this technique is being offered as a clinical service. "The
old-fashioned form of karyotype analysis will disappear," Dr. Beaudet
predicts. "You can diagnose things with array CGH that you can’t
diagnose with karyotyping alone. And you would have to combine hundreds
of FISH tests at the same time to equal its power." He has been in
clinical genetics for 35 years, he says, and this has been "by far
the biggest revolution" he has seen. "In terms of changes in
lab methodology of direct clinical relevance, I don’t think it gets any
more impressive than this," he says.
Bassem Bejjani, MD, FACMG, medical director of the other U.S. laboratory
offering array CGH for clinical diagnosis of chromosomal abnormalities,
says array CGH will create "a new place" in cytogenetics. "FISH
will not be eliminated, but its role will be transformed," he says.
Dr. Bejjani, who is research professor at the Health Research and Education
Center at Washington State University, co-director of the molecular diagnostics
laboratory at Sacred Heart Medical Center, and co-founder (with Lisa G.
Shaffer, PhD, FACMG) and medical director of Signature Genomics Laboratory,
predicts that array CGH will often be the first-choice test for suspected
chromosomal abnormalities. "I already have two physician clients
who send specimens and ask for array CGH first," he says. "If
it is normal, then we do a standard chromosome study. In the future, I
think this is exactly what will happen."
Scientists at Quest Diagnostics-Nichols Institute, San Juan Capistrano,
Calif., are developing their own version of this technology. "As
our understanding of the human genome increases, we’re finding that the
frequency of variations to our genomic content is much higher than anticipated.
Array CGH is an ideal tool to detect and characterize these variations,"
says Mansoor Mohammed, PhD, director of the Department of Advanced Technologies
at Quest Diagnostics-Nichols Institute.
He notes that, in the clinical diagnostic setting, array CGH is useful
in two primary situations. First, when there is an unclear phenotypic
presentation, array CGH can be used as a first option to screen the entire
genome for aberrations that would otherwise be difficult to predict. Second,
when a child presents with a classic syndrome but traditional karyotyping
or FISH analysis reaches a roadblock, array CGH can often detect the underlying
cryptic abnormality. In addition, when a standard technique does suggest
a chromosomal abnormality, "you can bring in array CGH to confirm
and clarify the chromosomal abnormality at a significantly higher resolution,"
Dr. Mohammed says. He argues that array CGH will become a cost-saving
method either when used independently or in conjunction with chromosome
analysis, as opposed to having to rely on potentially several rounds of
sequential FISH tests.
One application in which array CGH will be especially helpful will be
in identifying abnormalities in regions adjacent to the telomeres (sometimes
called subtelomeric abnormalities). Clones for these regions have been
developed by David H. Ledbetter, PhD, director of the Division of Medical
Genetics at Emory University School of Medicine, in collaboration with
researchers in the United Kingdom. These clones have revealed abnormalities
in three percent to five percent of cases of children with developmental
delay or mental retardation who had normal banding analysis. "Telomere
FISH moved rapidly into clinical practice," says Dr. Ledbetter, "but
it is limited obviously by looking for cryptic or submicroscopic abnormalities
only in one part of the genome at a time." It would be helpful to
be able to look for very small deletions and duplications anywhere in
the karyotype. "Current array technology probably represents the
best chance to do that," Dr. Ledbetter says.
A commercial array for CGH is being developed at Spectral Genomics in
Houston, a spinoff from the Baylor College of Medicine. "This type
of technology, once approved for clinical diagnostic laboratories, will
start replacing FISH analyses, effectively offering hundreds or thousands
of FISH tests at the same time," says Robert Johnson, PhD, Spectral’s
CEO and president. Dr. Johnson also expects to see some displacement of
conventional karyotyping, but to a lesser degree than FISH.
"It’s going to be very interesting to see how this works out,"
says Jean Amos Wilson, PhD, scientific director of human genetics at Focus
Diagnostics, Cypress, Calif. "It’s almost like cystic fibrosis was
in 1992, when we knew we had to test for a lot of mutations but we didn’t
have good platforms to develop homebrew assays and there were no ASRs."
For a laboratory to develop array CGH in-house now requires an intensive
development program. (Signature and Baylor both design and make their
own arrays.) CGH has enormous potential significance," says Dr. Amos
Wilson. She notes that Dr. Bejjani’s laboratory has used it to detect
a case of Duchenne’s muscular dystrophy, which is often caused by deletions,
in a child too young to be symptomatic.
Consideration of array CGH begins with advantages. As location is to
real estate, resolution is to array CGH. "You miss a lot looking
at chromosomes under a microscope," Dr. Bejjani says. With G banding,
in the best hands resolution is three to five megabases (Mb), while resolution
for any region with a bacterial artificial chromosome is 70 to 90 kilobases
(Kb). "That is our experience, and that of others," Dr. Bejjani
says. Resolution of FISH is similar to that of array CGH, but here other
advantages of CGH come into play. "In FISH you use a single probe
and must have a strong suspicion of which probe to use," Dr. Bejjani
says. Array CGH is equivalent to using hundreds of FISH probes in parallel
on a sample. In addition, array CGH uses not metaphase chromosomes but
extracted DNA, so culture is not needed, allowing a shorter turnaround
time. Dr. Bejjani says that in practice he can deliver a result in less
than five days.
Before describing what array CGH is and how it works, Dr. Bejjani is
careful to distinguish what it is not. "CGH arrays have nothing to
do with gene expression microarrays," he says. CGH arrays are structural,
not functional—they look for DNA that is missing or duplicated,
not DNA that is expressed.
In a way, array CGH turns FISH upside down. In FISH, sample DNA is laid
down on a substrate and dye-labeled probes are layered over this chromosome
spread. Array CGH, on the other hand, starts with printing hundreds or
thousands of probes on a microscope slide. These probes are bacterial
artificial chromosomes, or BACs, which are sequences of cloned human DNA
120-180 Kb long that have been mapped to specific locations on specific
chromosomes. (Most of the current library of BACs was prepared for the
Human Genome Project.) To the array is added DNA from a patient (test
DNA) and DNA from an apparently normal person (reference or control DNA),
each labeled with a different dye. The essence of the experiment is that
the two DNA samples compete for hybridization to the BAC probes on the
slide—the "comparative" aspect of CGH.
When each DNA sample contains equal amounts of the DNA corresponding
to a particular probe, equal amounts of each prep will hybridize to that
probe. If the patient’s DNA has a deletion, the reference DNA will be
twice as likely to hybridize to BACs covering that region. For a duplication,
there will be excess hybridization of patient DNA to corresponding probes.
After hybridization, the array is read by a laser scanner and a computer
calculates color ratios and translates them into gain or loss of a chromosome
region, which can identify a clinical syndrome.
"Array CGH works for any chromosomal rearrangement that shows a
dosage abnormality," Dr. Bejjani says. This means anything that changes
the copy number of a DNA segment—microduplications, microdeletions,
and unbalanced translocations. It does not detect balanced translocations
or inversions because all the DNA is present—it is just rearranged.
"Whenever you have a change in gene dosage, you will have clinical
consequences," Dr. Bejjani says. "Unbalanced chromosomal abnormalities
make up the vast reservoir of our patients. Balanced abnormalities make
up only a small percentage."
Several details of array CGH are important. Each affected region is represented
by more than one BAC on the array that Signature developed. In the affected
region of Potocki-Shaffer syndrome on 11p11.2, for instance, Signature’s
array contains nine BACs. "In each patient," Dr. Bejjani says,
"for any one locus that deviates from baseline, the array results
should deviate for all BACs at that locus."
While Dr. Bejjani uses BACs as probes on his arrays, other groups use
synthetic oligonucleotides, or "oligos," which can be as small
as 45 base pairs in length, as compared with 100,000 base pairs for a
small BAC. In theory, oligos offer better resolution, since the longer
BACs may sometimes bridge the affected region. Using multiple overlapping
BACs for each affected region minimizes this problem. Oligos, on the other
hand, have a higher noise-to-signal ratio, Dr. Bejjani says, and thus
are more difficult to interpret.
BACs are purchased from commercial sources and don’t always contain what
is stated on the label. All BACs must be tested in-house to see whether
the clone contains the DNA that it is supposed to contain—otherwise
it will mismap—and to ensure that it contains unique DNA, not repetitive
sequences, or it will hybridize to multiple chromosome sites.
Also, a BAC must not be from a polymorphic region of the genome. When
Dr. Bejjani’s laboratory tested a group of probes against the DNA of 50
apparently healthy individuals, many probes showed gain or loss. "We
decided that for diagnostics we want to avoid these regions," Dr.
Bejjani says. "Every time you find a polymorphism, you have to follow
up on it, which is costly."
In sum, out of 906 clones that Dr. Bejjani tested, 61 (seven percent)
mapped to the wrong region and 149 (17 percent) cross-hybridized to more
than one region. Only 589 (65 percent) turned out to be suitable for the
array. Once the array was designed and printed, it had to be validated
by running it against many samples with known abnormalities and known
normal controls (Bejjani BA, et al. Am
J Med Genet A. 2005;134:259-267). Dr. Bejjani emphasizes that
this is a targeted array—one in which every BAC corresponds to a
genetic locus implicated in a congenital disorder. Arrays can be made
that cover the entire genome, but with that type of array many results
will not be interpretable in clinical terms.
At Signature, each array CGH analysis includes a male-female comparison—normal
DNA of the opposite gender from the test sample—as an internal control.
Between this pair of samples, there will always be a gene dosage difference
for the X chromosome. If a run shows no difference between the test and
reference samples, but does have the expected difference between the male
and female samples, that shows that the lack of difference is not due
to a technical deficiency in the run.
Each patient dataset is actually made up of two experiments on the Signature
platform—one in which the test and reference DNA samples each have
a different dye color and a second in which the dyes are switched. Both
experiments are plotted on a single graph and should show the exact inverse
of each other (Related article: DNA samples).
For technical reasons, data are plotted as log 2, rather than arithmetically.
Signature’s first array contained 831 BACs covering 126 loci. Dr. Bejjani’s
laboratory is developing a new array with greater coverage of the subtelomeric
and pericentromeric regions. Signature first offered array CGH as a clinical
service in March 2004. "In the first couple of months we had only
a handful of cases," Dr. Bejjani says. "Now we are doing 65
to 70 cases a week. We started with three employees, now we have 20."
To accommodate the increasing demand, Signature is moving toward automation.
"This technology allows you to automate more easily than conventional
cytogenetics," Dr. Bejjani says. "It is not as limited by human
skills and training."
Dr. Beaudet at Baylor also uses BAC arrays, but says, "There is
plenty of literature to indicate that oligo arrays have enormous attraction.
Maybe they will be the final format for this technology." They can
print BAC arrays now cost-efficiently, he says: "We can print the
arrays and do a complete analysis for a little over $1,000." If they
switched to oligo arrays, simply purchasing the arrays themselves would
cost $1,000 or more, Dr. Beaudet says. Affymetrix, which holds the right
to printing oligo arrays, "is unwilling to negotiate a license,"
according to Dr. Beaudet. Nonetheless, he believes that oligo technology
will eventually predominate. "Signal-to-noise problems can be overcome,"
Just as Dr. Bejjani did, Dr. Beaudet had to validate his array in-house
(Cheung SW, et al. Genet
Med. 2005;7:422-432). Regarding polymorphisms, or benign variants,
Dr. Beaudet says, "We have tried to weed out those clones. If we
do use one, we check the [healthy] parents to see if the same gain or
loss is present in their genome," which would indicate that it is
probably not the cause of the child’s problem.
"We are just beginning to offer array CGH on a prenatal basis,"
Dr. Beaudet says. "It has tremendous attraction. It can diagnose
a number of disorders that would not otherwise be diagnosed." Examples
include diGeorge syndrome, Angelman syndrome, and Prader-Willi syndrome.
"For someone having an amniocentesis, array CGH becomes a possibility,"
Dr. Beaudet says. "Should we be offering all women an invasive test
such as amniocentesis?" he asks. "Some people are arguing that
the answer is yes" (Caughey AB, et al. Obstet
Noninvasive alternatives include finding fetal cells in maternal circulation,
isolating fetal cells with a cervical cell collection, and isolating fetal
DNA from maternal plasma. It is unlikely that prolonged presence of cells
or DNA from previous pregnancies would be a problem with the latter two
methods, according to Dr. Beaudet. "In five to 10 years we may be
able to do array CGH on noninvasive samples," he says. "That
would be a huge revolution in genetic diagnosis." When they counsel
parents-to-be now they say that there is "some merit" to doing
amniocentesis in all pregnant women and, if parents elect an amniocentesis,
they offer array CGH as an additional test.
Array CGH will definitely remain a sendout for a while, says Dr. Amos
Wilson. Laboratories desiring array CGH analysis will be sending samples
to either the Signature or Baylor laboratories. They would have a choice
of how to proceed, Dr. Amos Wilson explains. "If the clinician suspects
an unbalanced chromosome rearrangement, the choices are not doing routine
cytogenetics at all, or maybe doing routine cytogenetics to look for large
alterations, then sending a sample to one of these laboratories."
For now, array CGH will be secondary to routine cytogenetics but will
allow medical geneticists to skip FISH, in Dr. Amos Wilson’s view. "CGH
could be considered superFISH," she says. "You do FISH because
you suspect a specific chromosome alteration. With array CGH you can look
at the whole genome when you don’t have a suspected target." Referring
laboratories will also have to make a financial decision, she says. "Laboratories
make no money if they send out."
A case studied by Quest’s Dr. Mohammed and Shelley Gunn, MD, PhD, illustrates
the power of array CGH. A child presented with a classic phenotype consistent
with what would be expected as a consequence of a large deletion of the
distal portion of 18q—facial abnormalities, malformation of limb
development, and abnormalities of white brain matter. Yet chromosome analysis
appeared normal. "Large regions of 18q can be deleted and still be
consistent with life," Dr. Mohammed says, "so this anomaly should
have been easily seen on chromosome analysis." After years of chromosome
and FISH analyses, the case reached Dr. Mohammed’s laboratory. FISH probes
confirmed a large deletion of 18q. Array CGH uncovered the basis of the
abnormality: the large (approximately 7 Mb) deletion on 18q was balanced
by a concomitant duplication of material from chromosome 4q in the segment
of 18q from which DNA had been deleted. This case highlights the need
for "innovative molecular cytogenetic techniques," the authors
wrote (Gunn SR, et al. Am
J Med Genet A. 2003;120:127-135). "Within the year,"
Dr. Mohammed says, "a colleague in France, Dr. Anne Moncla, who had
a patient with a classic presentation of 18q deletion syndrome that could
not be confirmed by chromosome studies, used our array CGH approach and
found that her patient in fact had the exact breakpoints and unbalanced
translocation as our child" (Am J Med Genet A. 2004;131:131).
The odds of uncovering this finding by any other approach would have been
"incredibly small," he adds.
Neither the Baylor laboratory nor Signature Genomics sells their arrays.
However, at least two companies are developing commercial CGH arrays.
At Abbott Molecular Inc. (formerly Abbott/Vysis), a product for research
use only is available, says medical director Timothy T. Stenzel, MD, PhD,
FACMG. are targeting the research market right now," Dr. Stenzel
says. "But we do have a plan to offer array-based CGH for diagnostic
purposes in the future and would like to get FDA regulatory clearance."
Spectral Genomics has two products for research use, Dr. Johnson says.
One is a chip with more than 2,600 BAC clones spaced roughly 1 Mb apart
that gives a whole-genome view of changes in copy number. The second is
a chip with a much smaller number of BACs that is targeted to constitutional
syndromes. In its current research version, it is designed to pick up
42 congenital disorders as well as 41 subtelomeres. Spectral initially
approached the FDA in October 2003 with the 1-Mb chip, but the agency
expressed a desire for a more focused device, Dr. Johnson says. "So
we honed in on constitutional syndromes." Dr. Johnson notes that
there is a body of literature describing which chromosomes and which areas
of those chromosomes show changes corresponding to specific constitutional
syndromes. As with Dr. Bejjani’s and Dr. Beaudet’s diagnostic arrays,
a change in a specific spot correlates with a specific syndrome. Spectral
has submitted a pre-IDE that is under review. It intends to go for a 510(k)
designation, Dr. Johnson says.
In Dr. Beaudet’s view, the "FDA is currently a potential obstacle
to this technology." The FDA appears inclined to think about a relatively
limited number of clones, he says, whereas people are already using arrays
with 800 to 1,000 BACs and are likely to go higher.
Future additions to clinical diagnostic arrays for CGH will include more
BACs covering subtelomeric regions, which Dr. Ledbetter prefers to call
"unique telomere clones." He began working more than 10 years
ago—before the Human Genome Project’s physical map was as complete
as it is now—to identify clones for DNA regions that are unique
to each chromosome end and as close to the end as possible. Abnormalities
near the ends of chromosomes underlie many developmental anomalies, particularly
mental retardation syndromes. However, Dr. Ledbetter says, "The very
end of each human chromosome arm contains repeat sequences common to many
chromosomes." To get diagnostically useful clones that are specific
or unique required a lot of physical mapping. Once such probes were obtained
and validated, they were incorporated into FISH kits and are now standard
of care in unexplained cases of developmental delay or mental retardation
with normal banding analysis. Dr. Ledbetter says he was "surprised"
when the unique telomere clones were found to explain three percent to
five percent of such cases. "When we were first developing the probe
set and thinking about the frequency with which you might find telomere
imbalances below the resolution of karyotyping," he says, "I
thought one percent would be useful and clinically important." Finding
the much higher value actually makes these probes "clinically extremely
valuable," he says.
Another clinical value of the unique telomere probes is that parents
of children with telomere imbalances have been found to have a 50 percent
probability of being carriers of balanced telomere rearrangements. It
is known from studying couples with multiple miscarriages that a parent
with a balanced translocation can contribute a cytogenetically visible
unbalanced translocation to an offspring. "We have now discovered
that cryptic unbalanced rearrangements are also inherited from a balanced
and normal parent carrier," Dr. Ledbetter says. "That’s important
While array CGH sails smoothly along, ahead lies a potentially dangerous
iceberg—intellectual property conflicts. "We have a very strong
patent position in this area," says Dr. Stenzel of Abbott Molecular.
They have exclusively licensed the University of California’s fundamental
CGH patents, which Dr. Stenzel says are related both to CGH and array
CGH (Kallioniemi A, et al. Science.
1992;258:818-821), as well as UC’s array CGH patents and Deutsches Krebs
forschungszentrum’s matrix CGH patents out of the University of Heidelberg.
Laboratories and companies offering, or intending to offer, array CGH
have responded to this situation in various ways.
Dr. Beaudet says, "Affymetrix believes that they have patents of
relevance to CGH, and we are seeking a license from them." The Baylor
laboratory is in "the final stages" of negotiating a license
from Affymetrix for BAC arrays. "Eventually there will be a fight
or negotiation between Abbott Molecular and Affymetrix," Dr. Beaudet
predicts. "One possibility is that we will need a license from both
Dr. Bejjani is more cryptic about Signature’s approach. "We are
in discussion with a number of companies regarding licensing agreements,"
he says. Confidentiality agreements prevent him from being more specific.
Spectral’s work is covered by exclusive licenses from Baylor for crosslinking
DNA and attaching it to any surface and for the preparation and manufacture
of arrays, and by a license from Affymetrix that covers a large number
of patents on the ability to print arrays, Dr. Johnson says. Asked about
the UC’s fundamental CGH patent, he says, "Based on what we know
about the patent, that is not something we are concerned about."
Quest is taking a different approach and has developed a proprietary
method for array CGH. "The fundamental premise of current array CGH
approaches hinges upon using two colors to differentiate binding derived
from the test and reference samples respectively," Dr. Mohammed explains.
Quest’s innovation is to use only one color. The initial steps of standard
CGH are actually performed with no color, but each DNA sample is tagged
with a short unique DNA sequence. After hybridization, when the comparative
genomic step has been done, a fluorochrome is added to which is attached
the complement to the tag on the reference sample. Scanning at this point
gives the amount of reference sample hybridized. Then the same fluorochrome
is added, but this time it has attached to it the complement to the tag
on the test sample. Scanning this reaction gives the total signal. Subtracting
gives the test signal. Since these steps are fast, little time is added
to the procedure, Dr. Mohammed says.
Reimbursement is another bureaucratic hurdle. "It will be very important
to know that a laboratory can be reimbursed for this," says Dr. Amos
Wilson. "As with all molecular testing, as technology changes we
don’t have CPT codes that are specifically applicable."
Says Dr. Bejjani: "Right now we do institutional billing, insurance
billing, patient billing—the same as with other tests, such as FISH.
We are starting to make headway with reimbursement with some carriers."
He agrees that CPT codes are a problem. He uses CPT codes from other technologies
for individual steps, such as DNA preparation, hybridization, and interpretation
and bundles them, "as in the early days of FISH."
Dr. Beaudet says that at present the Baylor laboratory requires guaranteed
payment from hospital sendout laboratories. "I am very optimistic
about reimbursement within the next two years," he says.
Dr. Bejjani brings up one final consideration: Who should interpret array
"With this new technique, the conceptual line has been blurred between
cytogenetic conditions that are diagnosed with G band analysis and molecular
conditions that have been traditionally diagnosed with techniques such
as PCR and Southern blots," he says. Array CGH is both a cytogenetic
and a molecular technique. "I feel cytogeneticists need to learn
this technique and do the interpretation," Dr. Bejjani says. "For
chromosomal diagnosis we need someone who knows meiosis and who knows
which family members need to be tested. Cytogeneticists have been doing
this forever and are more attuned to the mechanisms of chromosomal abnormalities."
At the same time, he acknowledges, "All of this separation is artificial.
I think we are moving toward a unified world, in which cytogeneticists
are trained to know molecular and molecular biologists are trained to
know cytogenetics. Those who will navigate this field will be those who
William Check is a medical writer in Wilmette, Ill.