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Cytogenetics

Theory & Limitations

Chromosomal Microarray Analysis (CMA) represents the transition from “looking” at chromosomes (microscopy) to “measuring” them (molecular quantification). While G-banded karyotyping provides a low-resolution overview of the entire genome (~5–10 Mb resolution), CMA provides a high-resolution scan capable of detecting Copy Number Variants (CNVs) as small as 10–50 kilobases (kb). It is currently the first-tier diagnostic test for developmental delay, intellectual disability, and multiple congenital anomalies

Theory: Comparative Genomic Hybridization (aCGH)

The dominant platform for clinical microarray is Oligo-SNP array or aCGH. The fundamental principle is Competition and Ratio Analysis

  1. The Chip (The Array): The testing platform is a glass slide printed with hundreds of thousands (or millions) of microscopic DNA spots. Each spot contains a specific DNA sequence (oligonucleotide probe) representing a unique location in the human genome
  2. The Samples
    • Patient DNA: Extracted from the patient’s blood or tissue. Labeled with a fluorescent dye (e.g., Cyanine-3 / Green)
    • Reference DNA: A normal control DNA (pooled from healthy individuals). Labeled with a different dye (e.g., Cyanine-5 / Red)
  3. The Competition
    • The Patient (Green) and Reference (Red) DNA are mixed together in equal amounts and applied to the chip
    • They compete to hybridize (bind) to the probes on the glass
  4. The Ratio (The Result)
    • A laser scanner measures the fluorescence intensity of Red vs. Green at every single spot
    • Yellow (Equal): If the patient has 2 copies (Normal) and the Reference has 2 copies, the binding is equal (1:1 ratio). The spot appears Yellow. Result: Normal Copy Number.
    • Green (Gain): If the patient has 3 copies (Duplication/Trisomy) and the Reference has 2, the Patient DNA out-competes the Reference. There is more Green dye. Ratio > 1.0. Result: Duplication.
    • Red (Loss): If the patient has 1 copy (Deletion/Monosomy) and the Reference has 2, the Reference DNA wins. There is more Red dye. Ratio < 1.0. Result: Deletion.

Theory: SNP Arrays (Single Nucleotide Polymorphism)

Modern clinical arrays combine aCGH probes (for copy number) with SNP probes

  • Mechanism: These probes detect the specific genotype (AA, AB, or BB) at thousands of polymorphic sites across the genome
  • Benefit: Unlike aCGH, which only detects relative quantity (Gains/Losses), SNP probes can detect Copy Neutral abnormalities
    • Uniparental Disomy (UPD): When a child inherits two copies of a chromosome from one parent and zero from the other. The copy number is normal (2 copies), so aCGH misses it. But SNP analysis reveals the genotype is entirely homozygous (AA or BB, no AB), identifying the defect
    • Loss of Heterozygosity (LOH): Regions of homozygosity (ROH) can indicate consanguinity (parents are related) or recessive disease risk

Limitations of Microarray

Despite its high resolution, CMA is not a perfect replacement for Karyotyping. It has distinct “blind spots” based on its methodology

  1. Balanced Rearrangements (The Invisible Translocation)
    • Limitation: CMA measures Quantity, not Structure
    • Scenario: If a patient has a balanced reciprocal translocation (e.g., t(11;22)), the total amount of DNA is normal (2 copies of everything). The pieces are just in the wrong places
    • Result: CMA will be Normal.: Karyotyping is still required to detect balanced rearrangements (which cause infertility or recurrent miscarriage)
  2. Low-Level Mosaicism
    • Limitation: Because DNA is extracted from millions of cells and ground up together, the result is an “average.”
    • Scenario: If a patient has mosaic Trisomy 8 in only 10% of their cells, the extra signal is diluted by the 90% normal DNA. The ratio shift is too subtle to trigger a “Duplication” call
    • Threshold: CMA typically requires 20–30% abnormal cells: to detect mosaicism, whereas FISH/Karyotype can detect lower levels (single cell visualization)
  3. Polyploidy (Triploidy 69,XXY)
    • Limitation: Standard aCGH normalizes the signal. If the entire genome is gained (3 copies of everything), the software interprets “3 vs 2” as the new baseline “Normal,” effectively masking the Triploidy
    • Correction: SNP probes can detect this (via abnormal allele patterns like AAB or ABB), but pure aCGH platforms miss it
  4. Variants of Uncertain Significance (VUS)
    • Limitation: We find things we don’t understand
    • Scenario: CMA frequently detects small CNVs (e.g., a 200kb deletion) that have never been seen before in literature. The lab cannot determine if this is the cause of the disease or a benign familial trait. This leads to anxiety and requires parental testing (Trio Analysis) to resolve
  5. Mechanism Agnostic
    • Limitation: CMA sees a “Gain,” but it doesn’t know how it happened
    • Scenario: A gain of 21q could be simple Trisomy 21 (Non-disjunction) or an Unbalanced Robertsonian Translocation (Inheritable). CMA reports “Gain of 21q,” but a Karyotype is needed to see the mechanism and determine recurrence risk for the parents