Array-CGH, Karyotype Analysis, and FISH

Although many laboratories are using cultured cells at this point, some studies are demonstrating the feasibility of relying on uncultured samples, and ultimately, this is the direction in which we're heading. Direct testing of fetal DNA will save time and give us rapid results.

The Limitations of Array-CGH

Unlike standard karyotyping, array-CGH cannot detect defects in which the total amount of chromosomal material is unchanged. The test cannot, for instance, detect balance rearrangements, such as balanced reciprocal translocations, balanced Robertsonian translocations, and inversions.

In a couple with multiple miscarriages, a karyotype is still the appropriate test to perform on the parents' blood because a balanced rearrangement is what you would be looking for. You would not request array-CGH because balanced rearrangements are not detectable with this technique. On the other hand, array-CGH could be very useful on the products of conception from a miscarriage because very small deletions and duplications could be found.

Array-CGH also cannot detect point mutations, or small changes in the genes, like those that cause hemophilia or sickle cell disease. It is designed to detect the syndromes caused by duplications or deletions of larger amounts of chromosomal material. And it will not detect abnormalities that are not covered by the array.

Chromosomal mosaicism, in which only some cells show a particular abnormality, may or may not be more readily detected by array-CGH than by standard techniques.

On one hand, mosaicism may be more readily detected with array-CGH than with standard karyotype analysis because abnormal cells often do not divide as well and may be lost during the culture process that is part of the standard karyotyping methodology. On the other hand, experts believe that array-CGH may not detect mosaicism below a certain level—below the level, some say, at which the abnormality affects fewer than 15%–30% of cells.

Array-CGH will also inevitably detect normal variants (benign duplications and deletions that are not associated with any abnormal phenotype). Some variants will be difficult to explain. This has been true for karyotyping as well, and just as we have in the past, we will want to minimize parents' anxiety over the unknowns.

When we find variants of uncertain significance, we will turn to the parents, checking their blood samples for the same losses or gains of chromosomal material.

The Near Future

The clinicians and cytogeneticists who are using and offering array-CGH are on a learning curve. Experts seem to have been successful in ensuring that the test works for the disorders that are covered; there is an enormous amount of information and data being shared by centers and labs on what variants are associated with the normal phenotype, and on other issues as well.

At Johns Hopkins University and the Kennedy Krieger Institute, we have postnatal experience to draw upon as we bring array-CGH into the prenatal arena. Of the children with developmental delay and dysmorphic features who have had array-CGH, we have been able to give a specific syndromic diagnosis to approximately 5%–8%, depending on the array platform we utilize. In about 12%, we have detected variants that we know—through parental testing and the use of databases—are normal. In a much smaller percentage (3.4%) of these children, we have found variants that we cannot yet explain.

Until we learn more, we plan to limit prenatal array-CGH to cases in which there is a known abnormality on ultrasound, rather than offer the test more broadly as a screening tool for chromosomal abnormalities in high-risk pregnancies. And although we are moving in the postnatal setting toward more of a whole-genome screening, we will use targeted arrays in the prenatal setting.

Within this context—that of ultrasound-detected anomalies and targeted arrays—we can expect that 5%–10% of tests will provide a clear diagnosis.

The question of whether array-CGH could replace a karyotype in prenatal testing is an interesting one. For now, there are too many questions and issues (mosaicism and normal variants, for instance) to do away with karyotyping. We believe the role of array-CGH is to enhance our current approaches to prenatal testing, and in this sense, it is an exciting development.

Figure A shows a hybridized array of >4,200 BAC clones; B, one area enlarged; C, plot for chromosome 1 based on fluorescence ratios (patient vs. control DNA) showing normal copy number. Courtesy Dr. Denise Batista

Prenatal Diagnosis

In our contemporary society, where women and their physicians continue to seek as much information as possible early in their pregnancies, the field of prenatal diagnosis has rapidly become a well-established and central part of obstetrics. Prenatal diagnosis performed in the first trimester has become common practice—a far cry from the days in the not-so-distant past when the ultimate outcome of the fetus was not learned until the day of delivery.