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Cytogenetics

Microarray

Chromosomal Microarray (CMA) is the modern gold standard for detecting Copy Number Variants (CNVs) - gains and losses of genetic material - at a resolution 100x higher than standard karyotyping. It “measures” the genome rather than visualizing it

Theory & Limitations

  • Theory (Competition)
    • aCGH: Patient DNA (Green) and Reference DNA (Red) compete to bind to probes on a chip. The ratio of Green:Red intensity determines the copy number
      • Equal (Yellow): Normal (2 copies)
      • Green Dominant: Gain/Duplication (3 copies)
      • Red Dominant: Loss/Deletion (1 copy)
    • SNP Array: Adds probes to detect Genotype (AA/AB/BB). Allows detection of Uniparental Disomy (UPD) and Loss of Heterozygosity (LOH) (copy-neutral defects that aCGH misses)
  • Limitations
    • Balanced Rearrangements: CMA counts total DNA. It cannot detect balanced translocations or inversions because there is no net gain or loss
    • Mosaicism: Low-level mosaicism (<20%) is often missed because the signal is diluted by normal cells
    • Mechanism: It sees a “Gain” but cannot tell if it is a free trisomy, a ring chromosome, or an insertion. Karyotyping is needed for structural context

Evaluate & Confirm Results

  • Evaluation (Analysis)
    • QC Check: Assess “noise” (DLRS). If too noisy, the data is invalid
    • Visualization: The Log2 Ratio plot shows gains (shift up) and losses (shift down). The SNP (Allele) plot confirms these calls (loss of heterozygosity track)
    • Interpretation: CNVs are filtered through databases (ClinGen, DGV)
      • Pathogenic: Matches known syndrome (e.g., DiGeorge)
      • Benign: Found in healthy populations
      • VUS: Insufficient evidence (requires family studies)
  • Confirmation
    • Significant findings are typically confirmed by an independent method like FISH: or qPCR to rule out artifacts
    • Parental Testing: Crucial for VUS cases. If a healthy parent has the same deletion, it is likely benign. If it is de novo (new in child), it is likely pathogenic
  • Reporting: Results are reported using ISCN coordinates (e.g., arr[GRCh37] 16p11.2(...)x1)