ABPath Molecular Genetic Pathology cheat sheet

DNA is a double helix with antiparallel strands; adenine pairs with thymine (2 H-bonds), guanine with cytosine (3 H-bonds).

The human genome contains ~3.1 billion base pairs; protein-coding regions comprise ~1.5% of the genome.

Chromatin is composed of nucleosomes: 147 bp of DNA wrapped around an octamer of histones (2 each of H2A, H2B, H3, H4).

The central dogma: DNA → RNA (transcription) → protein (translation); reverse transcription (RNA → DNA) occurs in retroviruses and retrotransposons.

Human cells contain two copies of nuclear DNA (diploid) and multiple copies of mitochondrial DNA (2–10 per mitochondrion, 100–1000 mitochondria per cell).

Gene structure includes a promoter, 5' UTR, exons, introns, 3' UTR, and regulatory elements; the TATA box is ~25–35 bp upstream of the transcription start site.

RNA types: mRNA (coding), tRNA (amino acid carrier), rRNA (ribosome component), snRNA (splicing), miRNA (gene silencing).

The genetic code is degenerate: 64 codons encode 20 amino acids and 3 stop codons (UAA, UAG, UGA); AUG is the start codon.

PCR requires template DNA, primers, dNTPs, thermostable DNA polymerase, and buffer; cycling involves denaturation (~95°C), annealing (~55–65°C), and extension (~72°C).

Sanger sequencing uses dideoxynucleotides (ddNTPs) to terminate chain elongation; capillary electrophoresis separates fragments by size with single-base resolution.

Next-generation sequencing (NGS) platforms perform massively parallel sequencing; key steps include library preparation, clonal amplification (bridge or emulsion), and sequencing by synthesis or ligation.

Quantitative PCR (qPCR) uses fluorescent dyes or probes (e.g., TaqMan) to measure amplicon accumulation in real time; Ct value is inversely proportional to initial template quantity.

Southern blot detects specific DNA sequences after restriction digest, gel electrophoresis, and hybridization; Northern blot detects RNA; Western blot detects protein.

Sanger sequencing has a limit of detection of ~15–20% variant allele frequency; NGS can detect variants at 1–5% with sufficient depth.

Multiplex ligation-dependent probe amplification (MLPA) quantifies exon-level copy number changes and is used for deletions/duplications.

Digital PCR partitions the reaction into thousands of droplets, enabling absolute quantification and detection of variants at 0.1–0.01% allele frequency.

Mendelian inheritance patterns: autosomal dominant (vertical transmission, 50% risk per child), autosomal recessive (25% risk per child of carrier parents), X-linked recessive (males affected, carrier females), X-linked dominant.

Hardy-Weinberg equilibrium: p² + 2pq + q² = 1, where p and q are allele frequencies; carrier frequency for autosomal recessive = 2pq.

Mutation types: missense (amino acid change), nonsense (premature stop), frameshift (insertion/deletion not multiple of 3), splice-site, silent, and regulatory.

HGVS nomenclature: c. for coding DNA, g. for genomic, m. for mitochondrial, p. for protein; e.g., c.76A>T (p.Lys26*).

Loss-of-function variants include nonsense, frameshift, and canonical splice-site mutations; gain-of-function often results from missense mutations.

Trinucleotide repeat expansion disorders: Huntington disease (CAG), fragile X syndrome (CGG), myotonic dystrophy (CTG); anticipation occurs with increasing repeat size.

Mitochondrial DNA is maternally inherited; heteroplasmy refers to the coexistence of mutant and wild-type mtDNA, with threshold effects on phenotype.

Imprinting disorders: Prader-Willi syndrome (loss of paternal 15q11-q13 expression) and Angelman syndrome (loss of maternal UBE3A expression).

CLIA '88 regulates clinical laboratories; CAP provides accreditation and proficiency testing for molecular genetic tests.

Quality control includes positive and negative controls with each run; for NGS, metrics include coverage depth, uniformity, and variant allele frequency thresholds.

Quality assurance encompasses pre-analytical (specimen handling), analytical (test performance), and post-analytical (reporting) phases.

Proficiency testing (PT) is required for many molecular tests; alternative performance assessment is used when PT is not available.

Ethical issues: informed consent for genetic testing, confidentiality of genetic information, duty to warn at-risk relatives, and incidental findings in genomic sequencing.

Legal frameworks: Genetic Information Nondiscrimination Act (GINA) prohibits discrimination in health insurance and employment based on genetic information.

Regulatory: FDA regulates in vitro diagnostic devices; laboratory-developed tests (LDTs) are under CLIA and increasing FDA oversight.

Quality improvement uses metrics such as turnaround time, specimen rejection rates, and error rates; root cause analysis for adverse events.

Cystic fibrosis: autosomal recessive, CFTR gene mutations (e.g., F508del); carrier frequency ~1/25 in Caucasians.

Duchenne muscular dystrophy: X-linked recessive, DMD gene deletions; Becker muscular dystrophy is milder with in-frame deletions.

Huntington disease: autosomal dominant, CAG repeat expansion in HTT; reduced penetrance at 36–39 repeats, full penetrance at ≥40.

Fragile X syndrome: X-linked dominant with reduced penetrance, CGG repeat expansion in FMR1; >200 repeats cause methylation and silencing.

Mitochondrial disorders: MELAS (m.3243A>G in MT-TL1), MERRF (m.8344A>G in MT-TK), Leber hereditary optic neuropathy (m.11778G>A in MT-ND4).

Neurofibromatosis type 1: autosomal dominant, NF1 mutations; café-au-lait spots, neurofibromas, Lisch nodules.

Marfan syndrome: autosomal dominant, FBN1 mutations; aortic root dilation, lens dislocation, tall stature.

Hereditary hemochromatosis: autosomal recessive, HFE C282Y homozygosity in most cases; iron overload.

Chronic myeloid leukemia (CML): t(9;22) BCR-ABL1 fusion; imatinib targets the ABL1 kinase; quantitative PCR monitors BCR-ABL1 transcript levels.

Non-small cell lung cancer: EGFR mutations (exon 19 deletions, L858R) predict response to EGFR tyrosine kinase inhibitors; ALK rearrangements predict response to ALK inhibitors.

Colorectal cancer: KRAS/NRAS mutations predict resistance to anti-EGFR antibodies (cetuximab, panitumumab); BRAF V600E indicates poor prognosis.

Breast cancer: HER2 amplification/overexpression predicts response to trastuzumab; BRCA1/2 mutations confer hereditary risk and PARP inhibitor sensitivity.

Myeloproliferative neoplasms: JAK2 V617F in polycythemia vera, essential thrombocythemia, and primary myelofibrosis; CALR mutations in essential thrombocythemia and myelofibrosis.

Acute myeloid leukemia: NPM1 mutations and FLT3-ITD have prognostic significance; PML-RARA in acute promyelocytic leukemia responds to ATRA.

Lymphomas: MYC rearrangement in Burkitt lymphoma; BCL2 rearrangement in follicular lymphoma; t(11;14) CCND1/IGH in mantle cell lymphoma.

Tumor mutational burden (TMB) and microsatellite instability (MSI) predict response to immune checkpoint inhibitors.

HIV viral load is measured by quantitative RT-PCR; lower limit of quantification varies by assay (e.g., 20 copies/mL).

Hepatitis C virus (HCV) genotyping guides treatment duration and agent selection; sustained virologic response (SVR) is defined as undetectable HCV RNA 12 weeks after therapy.

SARS-CoV-2 detection uses RT-PCR targeting multiple viral genes (e.g., N, ORF1ab); cycle threshold (Ct) values correlate inversely with viral load.

Bacterial identification by 16S rRNA gene sequencing; broad-range PCR followed by sequencing for culture-negative infections.

Methicillin-resistant Staphylococcus aureus (MRSA) detection by PCR for mecA gene.

Quantitative cytomegalovirus (CMV) and Epstein-Barr virus (EBV) PCR are used to monitor transplant patients and guide preemptive therapy.

Hepatitis B virus (HBV) DNA quantification monitors response to antiviral therapy; resistance mutations (e.g., YMDD motif) detected by sequencing.

Mycobacterium tuberculosis complex detection and rifampin resistance by PCR (e.g., GeneXpert MTB/RIF).

Short tandem repeat (STR) analysis is the standard for human identity testing; the FBI CODIS core loci include 20 STR markers plus amelogenin for sex determination.

HLA typing for transplantation: high-resolution typing of HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 by next-generation sequencing or sequence-specific oligonucleotide probes.

Chimerism testing after hematopoietic stem cell transplant uses STR analysis to quantify donor and recipient fractions.

Paternity testing: compare obligate alleles; a child's alleles must be inherited from the parents; exclusion probability >99.9%.

Forensic DNA analysis: low copy number (LCN) DNA requires increased PCR cycles and stringent contamination controls.

Immunologic diseases: HLA-B27 associated with ankylosing spondylitis; HLA-DQ2/DQ8 with celiac disease; HLA-DR4 with rheumatoid arthritis.

Primary immunodeficiencies: severe combined immunodeficiency (SCID) due to mutations in IL2RG (X-linked), ADA, or RAG1/2.

HLA-B57:01 screening prevents abacavir hypersensitivity; HLA-B15:02 predicts carbamazepine-induced Stevens-Johnson syndrome.

CYP2D6 ultrarapid metabolizers may have reduced efficacy of codeine (prodrug) and increased risk of toxicity; poor metabolizers have reduced conversion to morphine.

CYP2C19 poor metabolizers have reduced activation of clopidogrel; alternative antiplatelet therapy (e.g., prasugrel, ticagrelor) is recommended.

CYP2C9 and VKORC1 variants influence warfarin dose requirements; genotype-guided dosing algorithms are available.

Thiopurine methyltransferase (TPMT) and NUDT15 deficiency increase risk of thiopurine (azathioprine, mercaptopurine) toxicity; dose reduction or alternative agents.

HLA-B*57:01 is strongly associated with abacavir hypersensitivity; screening is recommended before initiating abacavir.

Dihydropyrimidine dehydrogenase (DPYD) deficiency increases risk of severe fluorouracil toxicity; genotype-guided dose reduction.

UGT1A1*28 polymorphism increases irinotecan toxicity (neutropenia, diarrhea) due to reduced glucuronidation of SN-38.

G6PD deficiency: avoid oxidative drugs (e.g., primaquine, dapsone, rasburicase) that cause hemolysis.

Variant annotation databases: ClinVar, dbSNP, gnomAD, COSMIC, and OMIM; ACMG/AMP guidelines standardize variant classification (pathogenic, likely pathogenic, VUS, likely benign, benign).

Bioinformatics pipeline for NGS: base calling → alignment (e.g., BWA) → variant calling (e.g., GATK) → annotation (e.g., ANNOVAR, VEP) → filtering and interpretation.

FASTQ format stores sequence and quality scores (Phred); BAM/SAM format stores aligned reads; VCF format stores variants.

Quality metrics: Q30 (base call accuracy 99.9%), coverage depth (e.g., ≥30x for germline, ≥500x for somatic), and uniformity.

Human genome reference builds: GRCh37/hg19 and GRCh38/hg38; variants must be reported with the genome build and transcript reference.

Clinical decision support: electronic health record (EHR) integration of genomic results, computerized provider order entry (CPOE) with genetic test ordering.

Data security: HIPAA privacy rule protects protected health information (PHI); genetic data requires encryption and access controls.

Laboratory information management system (LIMS) tracks specimens, results, and quality metrics; interfaces with instruments and EHR.