Chromosomal Microarray Analysis (CMA) is a high-resolution genetic test that examines the entire human genome for subtle structural changes in the chromosomes. This technique identifies missing or extra pieces of genetic material, termed copy number variations (CNVs). CMA provides a detailed look, detecting submicroscopic imbalances associated with various health conditions. The test works by performing a direct, side-by-side comparison between a patient’s DNA and a normal reference DNA sample.
Clinical Applications
A physician may recommend CMA when standard testing cannot resolve a suspected genetic cause for a patient’s health concerns. CMA is utilized as a first-line diagnostic tool for children presenting with unexplained developmental delay or intellectual disability. It is also routinely ordered for individuals diagnosed with autism spectrum disorder, as many cases are linked to identifiable genomic changes.
CMA is particularly useful in cases involving multiple congenital anomalies, suggesting an underlying chromosomal cause. Furthermore, it plays a role in prenatal diagnosis when an ultrasound reveals structural anomalies in the developing fetus. Identifying a pathogenic CNV provides a definitive genetic explanation for a wide range of neurodevelopmental and physical issues.
Sample Preparation and DNA Labeling
The process begins with obtaining a biological sample, which is typically blood, amniotic fluid, or chorionic villus tissue. The first laboratory step involves extracting and purifying the DNA from the collected cells. This isolated DNA is then fragmented into smaller pieces to prepare it for analysis on the microarray chip.
Following fragmentation, the patient’s DNA undergoes differential fluorescent labeling. The patient’s DNA fragments are chemically tagged with one type of fluorescent dye, such as a green-emitting dye (Cy3). Separately, a chromosomally normal control DNA sample is identically fragmented and labeled with a different colored dye, such as a red-emitting dye (Cy5). This dual-color labeling is the foundation of the comparative CMA technique.
This differential labeling allows the simultaneous analysis of both the patient and control samples on the same microarray slide. Labeling the two DNA samples with distinct colors allows the test to directly compare the relative amounts of each DNA sample that bind to specific locations. The equal fragmentation and precise labeling ensure that any resulting color difference is due to a difference in DNA quantity.
The Array Hybridization and Scanning
The next step involves the microarray chip, a glass slide covered with thousands of microscopic spots. Each spot contains a specific, short sequence of single-stranded DNA known as a probe, designed to bind to a known region of the human genome. These probes are arranged to cover the entire set of chromosomes at high resolution.
The labeled patient and reference DNA are mixed and applied to the microarray chip. The chip is placed in a hybridization oven, allowing the DNA fragments from both samples to competitively bind (hybridize) to their complementary probes. This competition is crucial because the amount of patient and reference DNA that binds to a probe directly reflects the copy number of that genomic region in the patient.
After hybridization, the slide is washed to remove unbound DNA fragments. The microarray is then loaded into a specialized scanner that uses lasers to excite the fluorescent dyes on the bound DNA. The scanner measures the intensity of the light emitted by the two different dyes at every probe location. This converts the binding event into a quantifiable ratio of red-to-green light.
The scanner’s output is an image where each spot is assigned a color representing the ratio of the two fluorescent signals. A yellow spot indicates equal binding of red and green-labeled DNA, suggesting a normal copy number of two for that location. Conversely, a spot appearing predominantly red or green signals an imbalance, which is evidence of a copy number variation.
Interpreting Copy Number Variations
The raw data collected from the scanner is a set of fluorescence intensity ratios processed by specialized bioinformatic software. This software plots the signal ratios across the entire genome, creating a visualization that highlights deviations from the expected two copies. The resulting data points identify Copy Number Variations (CNVs), defined as regions where the patient’s DNA signal is significantly higher or lower than the reference signal.
A ratio greater than one at a specific location indicates a duplication, meaning the patient has extra copies of that DNA segment. Conversely, a ratio less than one signals a deletion, showing that a piece of the chromosome is missing. These CNVs are classified based on their size, the genes they encompass, and whether they have been previously reported in patient databases.
A geneticist reviews the flagged CNVs, categorizing them as pathogenic (disease-causing), benign (harmless variation), or a variant of uncertain significance (VUS). The VUS classification is applied when the CNV is novel or its clinical impact is not fully established, often requiring further investigation or parental testing. This final step links the detected genomic changes to the patient’s clinical presentation to provide a meaningful diagnosis.