Molecular Detection of Genetic, Inherited, and Neoplastic Disease
Inherited disease
Variants follow HGVS nomenclature (c. for coding DNA, p. for protein). Classification uses the five-tier ACMG/AMP framework: pathogenic, likely pathogenic, variant of uncertain significance, likely benign, benign, weighing population frequency, computational prediction, functional evidence, segregation, and de novo status. Know inheritance patterns (autosomal dominant, autosomal recessive, X-linked, mitochondrial), imprinting (Prader-Willi versus Angelman), repeat expansions (Huntington disease, fragile X), and screening pathways: carrier screening, cell-free DNA prenatal screening, chorionic villus sampling and amniocentesis, and preimplantation testing. Pharmacogenomic examples include CYP2D6, CYP2C19, TPMT/NUDT15, DPYD, and HLA-B*57:01.
Neoplastic disease
Separate germline from somatic testing. Somatic panels detect hotspot mutations, copy-number changes, gene fusions (BCR::ABL1, PML::RARA, EML4::ALK), microsatellite instability, tumor mutational burden, and homologous recombination deficiency. Liquid biopsy analyzes circulating tumor DNA; minimal residual disease testing tracks clonal burden after therapy.
Traps
Never report a variant of uncertain significance as causative. Tumor purity shapes variant allele frequency: a heterozygous somatic variant gives VAF roughly half the tumor cell fraction. FFPE causes deamination artifacts (C:G to T:A) at low VAF, and clonal hematopoiesis confounds plasma testing. Mosaicism may fall below detection limits.
Rule of thumb
Expected VAF ≈ 0.5 × tumor fraction for a heterozygous clonal variant. A VAF of 25% in a sample with 50% tumor cellularity fits a heterozygous clonal mutation; a VAF of 2% in a 60% tumor sample suggests subclonality or artifact.