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Chromosomal Microarray Testing

In Patients with Development Delay, Autism or other Congenital Anomalies


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Published: September 2011

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Chromosomal microarray testing is recommended as the first-tier test to detecßt chromosomal imbalances in persons with developmental delay/intellectual disability, autism spectrum disorders, or multiple congenital anomalies. Interpretation of test results is a complex process and detailed clinical information and genetic consultation are helpful for accurate interpretation.

Presenters: Erik Thorland, PhD

Presenters: Karen Wain, MS

  • Genetic Counselor in the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, Minnesota

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Our presenters for this program are Dr. Erik Thorland, Director of the Mayo Clinic Cytogenetics Laboratory and Karen Wain, Genetic Counselor in the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, MN. Dr. Thorland will describe the technical aspects of chromosomal microarray testing and Karen will discuss microarray testing from the clinical perspective, particularly current recommendations for when to order testing and how to discuss testing and test results with your patient.

After viewing the Hot Topic, we invite you to participate in Beyond Hot Topic. This question and answer session will be posted online approximately 1 month after the Hot Topic presentation is posted. You can submit a question for Dr. Thorland or Karen at the end of the presentation. Alternatively, you can submit a question by selecting the Beyond Hot Topic link on the Hot Topic page. Thank you, Dr. Thorland and Karen.

History of Cytogenetic Testing

G-banded chromosome studies have been the workhorse of cytogenetics laboratories for over 40 years. The major advantage of this technique is that it presents a picture of the entire genome in a single assay. However, the major disadvantage of this technique is that it is very subjective and has a relatively low resolution such that deletions or duplications of genomic material below approximately 5 million base pairs or 5 megabases are generally not visible. Chromosome studies detect a rearrangement in approximately 4% of patients with developmental delay, autism, or multiple congenital anomalies.

Fluorescence in situ hybridization (FISH) testing came into use in clinical cytogenetics laboratories in the 1990s. The advantage of this technique is that it has relatively high resolution and can see deletions down to approximately 100 kilobases or 100 KB. However, the disadvantage is that you need to know where in the genome to look based on clinical findings. For example, historically a patient with clinical features of DiGeorge syndrome would get a FISH test for the region of chromosome 22 where deletions in this syndrome are common (as is shown in this example). However, chromosomal rearrangements elsewhere in the genome can also produce clinical features that are similar to DiGeorge syndrome. Therefore, the ultimate cytogenetic test would be able to examine the entire genome like a chromosome study, but do it at high resolution like a FISH study. This is where chromosomal microarray testing comes in.

Chromosomal Microarray Testing

There is more than 1 type of chromosomal microarray testing. At Mayo we use a technique called array comparative genomic hybridization or array CGH. The current array platform that we use in the clinical lab has approximately 50 fold higher resolution than a chromosome study and in the same population of patients, this technique detects chromosomal abnormalities in 15 to 20% of patients as opposed to 4%. I’ll go through the technique briefly. DNA is extracted from both a patient and a control. Both patient and control DNA are labeled with different fluorescent dyes, mixed, and cohybridized to the chip containing arrayed oligonucleotide probes. Following approximately 24-hour hybridization and washing steps, the arrays are scanned at approximately 2 micron resolution and the data are plotted on a log2 scale as shown on the next slide.

So this is an example of our current array that is in use clinically in the laboratory. There are 4 blocks on the array each representing a hybridization site for a single patient so we can analyze 4 different patients on the same microarray. Each of those blocks contains approximately 180,000 individual oligonucleotide probes and you can see individual differences in each spot on this array in various colors of red and green.

Chromosomal Microarray Data

This is what typical array data looks like once it’s plotted. Each probe is plotted independently and probes that have equal copy number between the patient and control DNA specimens line up on the zero line. Probes that are deleted should move to -1 as shown on the plot on the left which demonstrates a patient with a 13q deletion and probes that are duplicated should move to approximately 0.58 as is shown on the right which demonstrates a patient with a 14q duplication. The precise genomic position of each probe is known so the start and stop points or what we call the coordinates of each deletion and duplication can be described very accurately. Based on these coordinates, we can determine precisely what genes are contained within those intervals and make clinical judgments on the effects of deletions or extra copies of those genes.

180K Oligonucleotide Microarray

This slide shows a schematic representation of how our current clinical chip is designed. This chip has 180,000 independent probe locations. Most of these probes are evenly distributed across the genome at approximately 25,000 base pair or kilobase probe spacing. Since we need 5 consecutive probes showing the same deviation pattern designating either copy number loss or gain, our functional resolution is approximately 100 kilobases anywhere in the genome. However, some regions of the genome we know to be clinically significant (such as specific genes or regions such as the subtelomeric regions designated in blue on this slide or the pericentromeric regions designated in red) so we have approximately 500 targeted regions with additional probes at approximately 5 kilobase intervals giving us a functional resolution of 20 kilobases in those regions.

Limitations of Chromosomal Microarray

Every assay, of course, has limitations. The limitations of chromosomal microarray testing are that truly balanced rearrangements such as balanced translocations or inversions are not detectable with this assay-only unbalanced rearrangements are detectable. The other major disadvantage of chromosomal microarray testing is that it does not provide information on the structural nature of an imbalance. This type of information requires a second method and typically we utilize FISH testing to visualize the exact locations of deletions and duplications. This is very useful for defining unbalanced translocations and also differentiating between tandem duplications and insertional translocations.

This slide demonstrates an insertional translocation. The green probes are the centromere of chromosome 12 and the red probes are on distal chromosome 12q and you can see that there is an additional set of red signals and this actually is an insertional translocation into chromosome 2. This is a relatively rare type of rearrangement but it illustrates very well the utility of FISH testing to follow up what we would see on the array as a gain of multiple probes.

Human Copy Number Variation (CNV)

In humans, there is quite a bit of copy number variation, so advances in microarray technology have led to the discovery of widespread copy number variation in normal individuals. Much of this variation is attributed to common copy number polymorphisms. However, a small proportion of this variation is rare or novel, making interpretation of pathogenic significance difficult.

Interpretation of Results

So, on a typical 180k array that is run on a patient, we will see approximately 20 copy number changes per individual. Each of these copy number variations is assigned to 1 of 5 different categories: pathogenic, likely pathogenic, uncertain, likely benign, or benign. Thankfully most of these copy number changes can easily be assigned to either benign or pathogenic categories. However, a small subset of them have to have further interpretation. For copy number changes where there is a bit of uncertainty, we will classify them into either the likely pathogenic based on the size or the number of genes in the region or likely benign if there are very few or a small region or those that are truly uncertain we will actually sign out an equivocal result. So any CNVs of clinical concern are reported and discussed in the interpretive report.

When to Order a Microarray?

And now Karen Wain will discuss microarray testing from the clinical perspective, particularly current recommendations for when it is appropriate to order testing and what should be included when discussing testing with your patient.

Hello. Recently published clinical recommendations regarding chromosomal microarray testing are now available from the American College of Medical Genetics. These state that chromosomal microarray testing is a recommended first-line test for the initial postnatal evaluation of individuals with multiple congenital anomalies (particularly if not specific to a well-delineated syndrome), apparently nonsyndromic developmental delay or intellectual disability, or an autism spectrum disorder.

However, a standard karyotype is still the appropriate test for couples with multiple miscarriages or infertility, since balanced rearrangements which predispose to fertility problems would not be detected by microarray. Also, if a sex chromosome anomaly is suspected due to clinical features, a standard karyotype is recommended due to the complexities of possible mosaicism, particularly for suspected Turner syndrome.

Additional ACMG Recommendations

The ACMG recommendations also state that further studies are needed to determine the utility of chromosomal microarray for individuals with growth retardation, speech delay, and other indications, although we do certainly see testing ordered for these indications and, in our opinion, it is appropriate to consider testing in these clinical scenarios. Additionally, all results should be confirmed by a second test method, such as FISH analysis, parental evaluation is often necessary to interpret test results, and a clinical genetic evaluation and counseling is appropriate.

Pretest Counseling

In the next few slides I would like to run through some basic components of a genetic counseling session involving pre- and post-test counseling around microarray testing.

Pretest counseling for microarray testing is quite complex and requires some time with a family to explain what the test is, why it is being ordered, and what the potential outcomes may be. An explanation of what testing can do is a good place to begin. Include a review of what chromosomes are, that people typically have 2 copies of each chromosome, and that we inherit 1 from each parent. Explain that sometimes people have missing or extra chromosomal material that impacts their development and that this is what the test is looking for. Using pictures is very helpful in this explanation.

Since it is always possible that a copy number change of uncertain significance will be found, it is important that families are aware of this possibility. This anticipatory guidance can be immensely helpful to families if they have to deal with a test result of uncertain significance. Describe the possible test results to them. A pathogenic change is one that we know would impact the child’s health and likely explain his or her problems. In discussing uncertain results, it is very helpful to explain that we all have some gains and loss of genetic material that is just part of normal variation and that parental testing is sometimes needed to help understand the significance of a child’s result. Sometimes, even with this information we may not sure. And finally a normal result, in which case there may be a need for further testing or evaluations. I’d also like to point out that if the clinician is aware of the potential of nonpaternity, the lab needs to be informed of this.

Another reason it is important to discuss possible parental testing up front is that some parents may be afraid of the possibility that they may have passed down a genetic cause for their child’s problems. They may feel guilty or distressed by this. By discussing this possibility from the beginning you are setting the stage for them to express their feelings and ask their questions about results later, and it is an opportunity to reinforce that they did not do anything to cause a chromosome anomaly. Also, it will help them begin to understand the difficult concepts of incomplete penetrance and variable expressivity if these need to be discussed later.

Occasionally we will detect an anomaly that is unrelated to a child’s problems (such as 47,XXX in a child with multiple congenital anomalies) or an anomaly which confers a health risk that is not related to the reason for testing (such as deletion of a tumor suppressor gene that may lead to an increased risk for a tumor, often in adulthood). A brief mention of this possibility may be helpful.

Post-Test Follow-Up

Post-test counseling regarding results should ideally be done in person rather than over the phone. Also, patients benefit from hearing complex information multiple times and presented in different ways, so even if you feel confident in your understanding of results and your ability to communicate them, the patient or family would likely benefit from an additional consultation with a genetics professional. The American College of Medical Genetics and the National Society of Genetic Counselors websites both have directories to help find the genetics clinic nearest you.

Discussing test results with a family should include an explanation of what type of anomaly was found, again using pictures if possible. Depending on the anomaly found, it may be appropriate to provide natural history information about a condition, if it is well-described. Similarly, discussing a particular gene involved may be appropriate if it is one that is known to lead to a particular condition or predisposition when deleted or duplicated. However, it is generally not useful to provide your patient with a long list of genes, many of which have little or no true data regarding the consequence of deletion or duplication in humans. If the test result is uncertain and parental testing is suggested, explain again how parental testing will help clarify the child's results, and be sure to reiterate that we all carry some deletions and duplication that are a part of normal genetic diversity.

Recurrence-risk information is critical and can be a highly complex discussion. This is a topic that a genetics professional should certainly be consulted on given the types of unique rearrangements and difficulties that incomplete penetrance and variable expressivity pose.

The type of anomaly impacts recurrence risk. For example, almost all reciprocal translocations are unique to a family and there is likely no empiric data available to calculate a risk number specific to a particular family’s rearrangement. Therefore, recurrence-risk estimates need to consider the parents’ pregnancy history and family history as well as the size of the regions potentially involved in the various possible unbalanced gametes and whether the literature contains reports of individuals born with imbalances of these regions. And for parents who carry a balanced insertional translocation there is a recurrence risk of 50% for each pregnancy.

For isolated or independent imbalances, recurrence risk information usually depends on whether the imbalance is inherited or is de novo. Generally the risk of a de novo imbalance is low, though not 0% due to possible gonadal mosaicism. If a parent carries an imbalance, each pregnancy would be at 50% risk for inheriting it. However, the actual risk of an abnormal phenotype depends on the likelihood that the imbalance is actually pathogenic as well as factors such as penetrance and the range of variable expressivity.

This information could potentially be overwhelming for some families. Take time to acknowledge how they are reacting to test results and encourage them to explore their reactions with you.

Additionally, offer support services when they are available. Sometimes families find that they are interested in contacting these services after some time has passed since the initial results disclosure. There are 2 advocacy/support organizations available for chromosome anomalies: Unique and Chromosome Disorder Outreach. Both welcome contact from families with microdeletion/microduplications found by microarray. Also, there may be groups available for specific syndromes depending on your patient’s test results.

The International Standards for Cytogenomic Arrays (ISCA) Consortium

Finally I’d like to briefly inform you of a world-wide effort to improve chromosomal microarray testing, through the International Standards for Cytogenomic Arrays Consortium (or ISCA). The ISCA Consortium is a collaboration of over 160 cytogenetics laboratories from around the world and Mayo Clinic has been highly involved since its conception.

The ISCA Consortium

The goals of this consortium include improving the overall clinical utility of microarray testing by providing evidence-based standards for array design, agreeing on standards for the interpretation of results, and creating a public database of test results with clinical information which will serve as a resource for laboratories, researchers, and clinicians. This database is called the ISCA Clinical CNV Database and contains data generated through routine clinical testing in ISCA member laboratories. All data is completely deidentified. It is contained at NCBI in the database for genomic structural variation (dbVar) and can currently be accessed via the ISCA website. Membership in the consortium is free and individual clinicians are encouraged to join.

Providing Clinical Information

As part of our effort to contribute quality phenotypic data to the ISCA database, a standard clinical information form was created. Ideally this would be completed for all patients for whom microarray testing is ordered. This information allows the laboratory to provide the best clinical interpretation of test results and is critical for maximizing the utility of the ISCA database. It is available online at

While it is generally accepted that there is little to no risk to participating in the database, patients may opt-out without impacting their clinical testing by notifying the laboratory. This can be done at the time testing is ordered by indicating so on the order form or clinical information form, or by contacting the laboratory by phone or fax after testing is completed.


We hope this has been a helpful introduction to microarray testing which is now recommended as a first-line test for individuals with developmental delay or intellectual disability, multiple congenital anomalies, or autism spectrum disorder. Our new array platform contains 180,000 oligonucleotide probes for genome-wide resolution of approximately 100 kilobases and 20 kilobases in targeted regions. And again comprehensive genetic counseling is recommended.

Mayo Clinic Cytogenetics Laboratory

And I would just like to leave you with the phone number for our genetic counselor on-call pager. This is the most direct way to reach us and please feel free to contact us with any questions. Thank you for your time and attention.