Laboratory Genetics and Genomics: Assay Choice, Interpretation, and Confirmation
Core concepts
The laboratory domain tests whether you can move fluently between cytogenetics, molecular testing, and biochemical genetics, and justify why one assay answers a given question.
- Resolution and scope. Karyotype detects balanced rearrangements, aneuploidy, and large structural changes (~5–10 Mb) but misses submicroscopic copy-number changes. CMA/FISH/MLPA detect deletions and duplications but are blind to balanced events. Sequencing detects point variants and small indels but poorly detects CNVs and repeat expansions. Know the blind spot of each method.
- Variant interpretation. Apply ACMG/AMP criteria (PVS1, PS1–4, PM1–6, PP1–5, BP1–7) and remember Bayesian logic: a single strong criterion plus supporting evidence is not automatically pathogenic; PVS1 requires the null variant to be in a gene where loss of function is the disease mechanism.
- VUS discipline. A VUS is not actionable for predictive testing of relatives; segregation and functional data are what reclassify it.
- Quality metrics. Know sensitivity, specificity, positive predictive value, and how prevalence changes PPV. Mosaicism detection depends on tissue, depth of coverage, and the proportion of abnormal cells.
Traps
Questions love the balanced translocation with a normal array (array cannot see it), the mosaic trisomy missed on blood but found on buccal/skin, and the variant that is pathogenic but in the wrong inheritance context (e.g., a heterozygous variant in a recessive gene).
Rule of thumb
Ask three questions in order: (1) What is the size/type of lesion I must detect? (2) Which tissue is most likely to carry it? (3) Does the result need orthogonal confirmation (e.g., repeat expansion by PCR/Southern, methylation by MS-MLPA)?
Worked example
A child with developmental delay and a de novo apparently balanced translocation on karyotype: CMA is normal. The correct next step is not to dismiss the finding - breakpoint disruption of a gene may be causal, so sequencing or breakpoint mapping may be warranted.