ABMGG Molecular Genetic Pathology cheat sheet

Chargaff rules: in duplex DNA A=T and G=C, so %G+%C = 100 - (%A+%T); GC content correlates with melting temperature.

Duplex Tm approximation: Tm ≈ 64.9 + 41×(G+C−16.4)/N for oligonucleotides; each 1% increase in GC content raises Tm roughly 0.4-0.9 °C in typical salt.

Watson-Crick pairing: A-T has 2 hydrogen bonds, G-C has 3; higher GC content confers greater thermal stability and harder strand separation.

DNA polymerase requires a free 3'-OH and extends 5'→3'; proofreading (3'→5' exonuclease) activity lowers error rate ~10-100 fold.

Nucleic acids absorb UV at 260 nm; A260/A280 ≈ 1.8 for pure DNA and ≈ 2.0 for pure RNA; lower ratios indicate protein contamination.

Reverse transcriptase synthesizes cDNA from RNA template and lacks proofreading, giving higher error rates than high-fidelity DNA polymerases.

Hybridization stringency increases with higher temperature, lower salt (monovalent cation), and higher formamide; mismatch discrimination improves under stringent conditions.

Restriction endonucleases cleave at palindromic recognition sites; methylation sensitivity and star activity are key assay design considerations.

PCR cycle: denaturation ~95 °C, annealing ~50-65 °C (typically 5 °C below primer Tm), extension ~72 °C; amplicon size is set by primer positions.

Primer design rules: 18-25 nt, 40-60% GC, avoid 3' complementarity (primer dimers/hairpins), and check for SNP sites under the 3' end.

Real-time PCR quantification: Ct is inversely proportional to starting template; a shift of 1 Ct ≈ 2-fold change (assuming 100% efficiency).

Efficiency E = 10^(−1/slope) − 1 from standard curve; acceptable qPCR efficiency is 90-110% (slope −3.1 to −3.6).

Sanger sequencing resolution: capillary electrophoresis with fluorescent ddNTP terminators; mixed peaks suggest heterozygosity, mosaicism, or contamination.

NGS: cluster generation, sequencing-by-synthesis, and base calling; read depth and mapping quality determine confident variant calling.

Allele-specific PCR/ARMS detects known point mutations but must control for false negatives from polymorphisms under primers.

Microarray CGH detects copy number changes (deletions/duplications) but not balanced rearrangements or point mutations.

CLIA requires laboratories to establish and document performance specifications for each test: accuracy, precision, analytical sensitivity, analytical specificity, reportable range, and reference interval.

Validation vs verification: laboratory-developed tests require full validation; FDA-cleared tests brought into the lab require verification of the manufacturer's performance specs.

Analytical sensitivity = limit of detection (LoD); analytical specificity = ability to detect only the intended target without cross-reactivity.

Proficiency testing (PT) must be performed for each regulated analyte; failures require corrective action and may trigger alternative assessment.

Quality control frequency: each run or per manufacturer instruction; positive and negative controls should flank every batch of patient specimens.

Contamination control: unidirectional workflow (pre-amplification → amplification → post-amplification), separate rooms, and UNG/dUTP systems for carryover prevention.

Turnaround time and reportable elements must include methodology, limitations, and interpretive guidance consistent with professional guidelines.

CAP accreditation requires documented competency assessment, equipment calibration, and biannual method performance review.

ACMG/AMP 2015 framework classifies variants as pathogenic, likely pathogenic, uncertain significance, likely benign, or benign using criteria codes (PVS1, PS1-4, PM1-6, PP1-5, BA1, BS1-4, BP1-7).

PVS1 applies to null variants (nonsense, frameshift, canonical splice site) in genes where loss of function is the established disease mechanism.

Population frequency: BA1 (stand-alone benign) uses high allele frequency in population databases (e.g., gnomAD) exceeding expected disease prevalence.

Autosomal recessive disease requires two pathogenic variants (in trans); carrier testing and phase determination matter for interpretation.

X-linked disorders: males hemizygous; females may be carriers or manifest due to skewed X-inactivation.

Variant nomenclature follows HGVS: c. for coding DNA, p. for protein, and reference transcript must be specified.

Mosaic variants may show allele fractions below 50% and require orthogonal confirmation (e.g., Sanger or different assay).

Penetrance and expressivity vary; reduced penetrance variants require careful family studies and segregation data.

Driver vs passenger mutations: drivers confer selective growth advantage and are recurrently mutated in specific tumor types; passengers are incidental.

Actionable targets: EGFR mutations (lung), BRAF V600E (melanoma/colorectal), KRAS G12C, BRCA1/2 (PARP inhibitors), HER2 amplification, and MSI-H/dMMR (immunotherapy).

Tumor mutational burden (TMB) is mutations per megabase; high TMB (typically ≥10 mut/Mb) may predict immunotherapy response.

Variant allele frequency (VAF) in tumor tissue reflects tumor purity and clonal architecture; low VAF may indicate subclonal or germline origin.

Liquid biopsy (ctDNA) enables noninvasive monitoring but has lower sensitivity at low tumor burden; false negatives are common in early-stage disease.

Germline vs somatic distinction requires paired normal tissue or careful VAF/annotation review; ~5-10% of cancer variants are germline.

Microsatellite instability (MSI) arises from mismatch repair deficiency (MLH1, MSH2, MSH6, PMS2) and is detected by PCR fragment analysis or NGS.

Fusion detection: RNA-based NGS or FISH for translocations (e.g., ALK, ROS1, RET) where DNA breakpoints are intronic and hard to capture.

BCR-ABL1 t(9;22) defines CML and a subset of ALL; quantitative RT-PCR (IS) monitors response and major molecular response (MMR, ≤0.1% IS).

JAK2 V617F is present in ~95% of polycythemia vera and ~50% of essential thrombocythemia/primary myelofibrosis; CALR and MPL mutations follow.

PML-RARA t(15;17) defines acute promyelocytic leukemia (APL) and predicts response to ATRA/arsenic; rapid molecular confirmation is critical.

FLT3-ITD and NPM1 mutations in AML carry prognostic significance; FLT3 inhibitors (midostaurin, gilteritinib) target ITD/TKD.

Clonality assays: immunoglobulin/T-cell receptor gene rearrangement PCR distinguishes reactive from neoplastic lymphoid proliferations.

TP53 mutations in MDS/AML confer adverse risk; complex karyotype and biallelic TP53 inactivation are high-risk features.

Minimal residual disease (MRD) by NGS or flow cytometry at 10^-4 to 10^-6 sensitivity predicts relapse in AML/ALL.

CLL: del(17p)/TP53 and del(11q)/ATM confer adverse prognosis; IGHV mutation status informs prognosis.

Nucleic acid amplification tests (NAATs) detect pathogen DNA/RNA with high sensitivity; viral load assays quantify and monitor response to therapy.

False positives from contamination require strict unidirectional workflow and negative controls; false negatives from inhibitors (heme, heparin) require internal controls.

Multiplex respiratory panels detect multiple viruses/bacteria in one reaction; result interpretation must consider colonization vs infection.

HIV-1 viral load (copies/mL or IU/mL) monitors antiretroviral therapy; target not detected differs from detected below limit of quantification.

HCV RNA detection confirms active infection; sustained virologic response (SVR) is undetectable RNA 12-24 weeks post-treatment.

Genotypic resistance testing (e.g., HIV pol, HCV NS5A) identifies mutations conferring drug resistance and guides regimen changes.

16S rRNA gene sequencing is used for bacterial identification and culture-negative infections; it cannot reliably resolve species within some genera.

Whole-genome sequencing (WGS) enables outbreak tracing and metagenomic pathogen discovery in culture-negative cases.

CYP2D6, CYP2C19, CYP2C9, TPMT, DPYD, and UGT1A1 are common pharmacogenes; star-allele nomenclature defines functional haplotypes.

CYP2C19 poor metabolizers have reduced clopidogrel activation; ultrarapid metabolizers may have altered response to proton pump inhibitors.

TPMT/NUDT15 deficiency increases risk of thiopurine (azathioprine, 6-MP) toxicity; genotyping guides dose reduction.

HLA-B57:01 predicts abacavir hypersensitivity; HLA-B15:02 predicts carbamazepine-induced Stevens-Johnson syndrome in at-risk populations.

Identity testing uses STR loci (typically 13-24 CODIS core loci); match probability and random match probability quantify evidentiary strength.

Chimerism monitoring after hematopoietic stem cell transplant uses STR or SNP panels to quantify donor/recipient fractions.

Maternal cell-free DNA (NIPT) screens for fetal aneuploidy; fetal fraction and confined placental mosaicism affect interpretation.

Cell-free DNA and circulating tumor DNA (ctDNA) applications require assay sensitivity and specificity validation for intended use.

Karyotype resolution is ~5-10 Mb; FISH detects targeted loci (~100 kb-1 Mb); chromosomal microarray (CMA) detects copy number changes at kilobase resolution.

Balanced translocations and inversions are invisible to CMA and require karyotype or FISH; CMA detects pathogenic CNVs in developmental disorders.

ISCN nomenclature standardizes cytogenetic descriptions (e.g., 46,XX,t(9;22)(q34;q11.2)).

Variant calling pipeline: alignment (BWA), variant calling (GATK), annotation (VEP), and filtering by quality, depth, and population frequency.

ACMG/AMP sequence variant interpretation uses population databases (gnomAD), in silico predictors (SIFT, PolyPhen-2, CADD), and segregation data.

Copy number calling from NGS uses read depth, split reads, and discordant pairs; orthogonal confirmation by CMA or MLPA is recommended.

Reference genome builds (GRCh37/hg19 vs GRCh38/hg38) must be consistent across pipeline components; liftover errors are a known pitfall.

Pharmacogenomic and Mendelian variant reporting must include transcript reference, HGVS nomenclature, and clinical significance classification.