What is the difference between karyotyping, microarray (Microarray Analysis) analysis, and full exome sequencing (Full Exome Sequencing) in genetic testing?

Differences Between Karyotyping, Microarray Analysis, and Full Exome Sequencing

Karyotyping, microarray analysis, and full exome sequencing are distinct genetic testing methodologies with different capabilities, resolutions, and clinical applications, with microarray offering significantly higher resolution than karyotyping but unable to detect balanced rearrangements, while exome sequencing provides the highest resolution by analyzing individual gene mutations.

Karyotyping

• Conventional cytogenetic analysis that examines chromosomes at the metaphase stage 1
• Detects both balanced and unbalanced structural abnormalities as well as numerical chromosomal abnormalities (aneuploidy) 1
• Limited resolution (approximately 5-10 Mb), with a diagnostic yield of approximately 3.7% in children with global developmental delay 1
• Useful for confirming specific diagnoses like Down syndrome or when a common aneuploidy is suspected 1
• Cannot detect submicroscopic deletions or duplications 1

Microarray Analysis (Chromosomal Microarray)

• High-resolution, whole-genome technique that detects copy number variations (CNVs) - deletions and duplications - at a resolution of 50-100 kb 2, 1
• Two main types:
  • Array Comparative Genomic Hybridization (aCGH): compares patient DNA with reference DNA using differential labeling 1
  • Single Nucleotide Polymorphism (SNP) arrays: only patient DNA is hybridized, compared against standard reference controls 1
• Higher diagnostic yield than karyotyping (approximately 10% with limited BAC arrays) 1
• Allows simultaneous analysis of hundreds or thousands of genomic loci 1
• Cannot detect balanced rearrangements (translocations, inversions), some ploidy changes, or point mutations 1
• SNP-based arrays can additionally detect absence of heterozygosity (AOH), which may indicate uniparental disomy 1
• May have limited ability to detect low-level mosaicism (10-30% depending on the abnormality type) 1

Advantages of Microarray Analysis:

• Can use any sample that yields DNA of sufficient quality 1
• Higher resolution than conventional cytogenetic analysis 1
• Objective biostatistical algorithms for data interpretation 1
• Ready interface with genome browsers and databases 1
• Recommended as first-line test for individuals with developmental delays, multiple anomalies, or autism spectrum disorders 1

Limitations of Microarray Analysis:

• Cannot detect balanced chromosomal rearrangements 1
• Limited ability to detect low-level mosaicism 1
• Cannot determine chromosomal mechanisms of genetic imbalance 1
• Cannot detect point mutations or gene expression changes 1
• May identify variants of uncertain significance requiring parental testing 1

Full Exome Sequencing

• Analyzes the protein-coding regions (exons) of all genes in the genome, which constitute approximately 1-2% of the entire genome 3
• Highest resolution of the three methods, capable of detecting single nucleotide variants (SNVs) and small insertions/deletions (indels) 3
• Particularly useful for diagnosing rare monogenic disorders, especially when the condition is genetically heterogeneous 3
• Diagnostic yield of 25-30% in unselected children with rare monogenic syndromes 3
• Cannot reliably detect large structural variants, copy number variations, or variants in non-coding regions 3
• More expensive and complex to interpret than karyotyping or microarray analysis 3

Clinical Applications and Test Selection

• Karyotyping remains useful for:

  • Suspected common aneuploidies (e.g., trisomy 21,18, or sex chromosome abnormalities) 1
  • Evaluation of balanced rearrangements 1
  • Family history of chromosomal rearrangement in phenotypically normal individuals 1
  • Cases of multiple miscarriages 1

• Microarray analysis is recommended for:

  • First-line test for individuals with multiple anomalies not specific to a well-delineated genetic syndrome 1
  • Apparently nonsyndromic developmental delay or intellectual disability 1
  • Autism spectrum disorders 1
  • Clarifying chromosome abnormalities detected by G-band analysis 1

• Full exome sequencing is indicated for:

  • Suspected monogenic disorders with genetic heterogeneity 3
  • Cases where karyotyping and microarray analysis have been negative but genetic etiology is still suspected 3
  • Identification of specific gene mutations for targeted therapy 3

Common Pitfalls to Avoid

• Ordering microarray when a rapid turnaround time is needed (e.g., STAT newborn analysis) - conventional karyotyping can be performed within 48 hours 1
• Using microarray as first-tier test when a common aneuploidy is suspected - conventional karyotyping or targeted FISH may be more cost-effective 1
• Failing to recognize that microarray cannot detect balanced rearrangements, which may be clinically significant 1
• Not following up unclear microarray results with parental testing to determine clinical significance 1
• Assuming that a negative result on any single testing modality excludes genetic etiology - an integrated approach may be necessary 3