Chromosomal Microarray

Definition and Concept

Chromosomal Microarray (CMA) is a high-resolution, genome-wide molecular cytogenetic technique used to detect submicroscopic chromosomal imbalances, specifically Copy Number Variants (CNVs) such as microdeletions and microduplications. It has largely replaced conventional G-banded karyotyping as the first-tier diagnostic test for specific pediatric neurodevelopmental presentations.

Types of Chromosomal Microarray

1. Array Comparative Genomic Hybridization (aCGH): Uses competitive hybridization of patient DNA and control DNA.
2. Single Nucleotide Polymorphism (SNP) Array: Uses non-competitive hybridization to detect both CNVs and allelic composition (homozygosity/heterozygosity).

Clinical Indications (Tier-1 Testing)

According to the American College of Medical Genetics and Genomics (ACMG) and American Academy of Pediatrics (AAP) guidelines, CMA is indicated as a first-line diagnostic test for:

• Unexplained Global Developmental Delay (GDD) or Intellectual Disability (ID).
• Autism Spectrum Disorder (ASD).
• Multiple Congenital Anomalies (MCA) not specific to a well-delineated genetic syndrome.
• Fetal anomalies detected on prenatal ultrasound.

Methodology of Array CGH

1. Extraction and Labeling: Patient DNA and reference (control) DNA are extracted. Patient DNA is labeled with one fluorophore (e.g., green), and reference DNA is labeled with a different fluorophore (e.g., red).
2. Hybridization: The labeled DNA samples are mixed in equal amounts and co-hybridized to a microarray slide containing thousands to millions of specific DNA probes representing known chromosomal regions across the genome.
3. Washing and Scanning: Unbound DNA is washed away. A laser scanner excites the fluorophores, and the fluorescence intensity of each spot is measured.
4. Data Analysis: The ratio of green to red fluorescence is calculated by specialized software.
   • Equal ratio (Yellow): Normal copy number (Neutral).
   • Higher patient fluorescence (Green): Duplication (Gain).
   • Higher reference fluorescence (Red): Deletion (Loss).

Advantages Over Conventional Karyotyping and FISH

• Higher Resolution: CMA can detect genomic alterations as small as 10 to 100 kilobases (kb), compared to the 5 to 10 Megabase (Mb) resolution limit of standard G-banded karyotyping.
• Genome-Wide Assessment: Unlike Fluorescence In Situ Hybridization (FISH), which requires prior clinical suspicion to select specific locus-targeted probes, CMA interrogates the entire genome simultaneously.
• Does Not Require Cultured Cells: CMA utilizes DNA extracted directly from blood, saliva, or tissue, bypassing the need for viable, actively dividing cells in metaphase. This significantly reduces turnaround time and failure rates.

Limitations and Diagnostic Challenges

• Inability to Detect Balanced Rearrangements: Cannot identify balanced translocations or inversions, as there is no net loss or gain of genetic material.
• Inability to Detect Sequence-Level Mutations: Does not detect single base-pair point mutations, small insertions/deletions, or trinucleotide repeat expansions (e.g., Fragile X syndrome).
• Low-Level Mosaicism: May miss mosaic chromosomal abnormalities if the abnormal cell line constitutes less than 15 to 20 percent of the sample.
• Variants of Uncertain Significance (VUS): Frequently identifies CNVs whose clinical significance is currently unknown, leading to significant parental anxiety and complex genetic counseling challenges. Parental testing is often required to determine if the VUS is de novo (more likely pathogenic) or inherited from a healthy parent (more likely benign).