AGMT cheat sheet

Sanger sequencing: dideoxy chain termination; capillary electrophoresis; gold standard for single-nucleotide variants and small indels; read length ~500–1000 bp; limit of detection ~15–20% variant allele fraction.

Next-generation sequencing (NGS): massively parallel; short-read platforms (Illumina) produce 75–300 bp reads; long-read platforms (PacBio, Oxford Nanopore) produce >10 kb reads; used for panels, exomes, genomes, RNA-seq.

PCR-based methods: allele-specific PCR, real-time PCR (qPCR), digital PCR (dPCR); dPCR partitions sample into thousands of droplets, enabling absolute quantification and detection of variant allele fractions as low as 0.01–0.1%.

Multiplex ligation-dependent probe amplification (MLPA): detects copy number changes (deletions/duplications) of exons; peak height ratios <0.7 or >1.3 suggest loss or gain; cannot detect balanced rearrangements or point mutations.

Chromosomal microarray (CMA): comparative genomic hybridization (aCGH) or SNP arrays; detects copy number variants (CNVs) and regions of homozygosity (ROH); resolution typically 10–100 kb; does not detect balanced translocations or low-level mosaicism (<10–20%).

Karyotyping: G-banded chromosomes at 400–550 band resolution; detects aneuploidy, large structural rearrangements (>5–10 Mb), and balanced translocations; requires dividing cells.

Fluorescence in situ hybridization (FISH): locus-specific probes; detects deletions, duplications, and translocations (e.g., BCR-ABL1); resolution ~100 kb–1 Mb; can be performed on interphase nuclei.

Methylation-specific PCR (MSP) and bisulfite sequencing: assess DNA methylation at CpG islands; used for imprinting disorders (e.g., Prader-Willi/Angelman, Beckwith-Wiedemann) and MLH1 promoter hypermethylation.

Expanded carrier screening (ECS): panels for autosomal recessive and X-linked conditions; residual risk after negative screen = prior risk × (1 – detection rate); example: CF carrier risk 1/25 × (1 – 0.90) = 1/250.

Central dogma: DNA → RNA → protein; transcription in nucleus, translation on ribosomes; post-translational modifications affect function.

Mutation types: missense (amino acid change), nonsense (premature stop), frameshift (insertion/deletion not multiple of 3), splice-site (affects splicing), silent (no amino acid change).

Loss-of-function (LOF) vs gain-of-function (GOF): LOF often recessive (haploinsufficiency) but can be dominant (dominant-negative); GOF typically dominant.

Penetrance: proportion of individuals with a pathogenic variant who exhibit the phenotype; age-dependent penetrance common in adult-onset disorders (e.g., BRCA1/2, Lynch syndrome).

Variable expressivity: same genotype produces different phenotypes among individuals; influenced by modifier genes, environment, and stochastic factors.

Genomic imprinting: parent-of-origin-specific gene expression; examples: 15q11-q13 (Prader-Willi/Angelman), 11p15.5 (Beckwith-Wiedemann/Silver-Russell).

Mitochondrial genetics: maternal inheritance; heteroplasmy (mixture of mutant and wild-type mtDNA); threshold effect determines phenotype; mtDNA mutations can cause MELAS, MERRF, LHON.

Trinucleotide repeat disorders: dynamic mutations; examples: Huntington disease (CAG in HTT), fragile X syndrome (CGG in FMR1), myotonic dystrophy (CTG in DMPK); anticipation (earlier onset/severe with expansions).

Human genome: ~3.1 Gb; ~20,000 protein-coding genes; ~1.5% coding; common variants (SNPs) >1% minor allele frequency; rare variants <1%.

Karyotype nomenclature (ISCN): 46,XX or 46,XY normal; examples: 47,+21 (trisomy 21), 45,X (Turner syndrome), 46,XY,t(9;22)(q34;q11.2) (Philadelphia chromosome).

Aneuploidy: abnormal chromosome number; trisomy (three copies) or monosomy (one copy); viable autosomal trisomies: 13, 18, 21; sex chromosome aneuploidies: 45,X; 47,XXY; 47,XYY; 47,XXX.

Structural rearrangements: deletions (del), duplications (dup), inversions (inv), translocations (t), ring chromosomes (r), isochromosomes (i); balanced rearrangements do not change copy number but may disrupt genes or cause unbalanced offspring.

Robertsonian translocation: fusion of two acrocentric chromosomes (13,14,15,21,22); carrier has 45 chromosomes; risk for unbalanced offspring (e.g., translocation Down syndrome).

Microdeletion/duplication syndromes: detected by CMA or FISH; examples: 22q11.2 deletion (DiGeorge), 15q11-q13 deletion (Prader-Willi/Angelman), 7q11.23 deletion (Williams), 5p deletion (cri-du-chat).

Mosaicism: presence of two or more cell lines; detected by karyotype (≥10–20 cells analyzed) or CMA (less sensitive for low-level); example: mosaic trisomy 21 (46,XX/47,XX,+21).

Chromosome instability syndromes: Fanconi anemia (DEB/MMC test), ataxia-telangiectasia, Bloom syndrome; increased spontaneous chromosome breakage.

Prenatal cytogenetics: chorionic villus sampling (CVS) at 10–13 weeks; amniocentesis at 15–20 weeks; cell culture required for karyotype; CMA can be done directly.

Autosomal dominant: affected individual has affected parent (unless de novo); 50% recurrence risk for each offspring; examples: Huntington disease, Marfan syndrome, familial hypercholesterolemia.

Autosomal recessive: both parents carriers (usually unaffected); 25% recurrence risk; consanguinity increases risk; examples: cystic fibrosis, sickle cell disease, Tay-Sachs disease.

X-linked recessive: males affected; females carriers (usually unaffected); no male-to-male transmission; 50% of sons of carrier female affected; examples: Duchenne muscular dystrophy, hemophilia A/B.

X-linked dominant: affected males transmit to all daughters (no sons); affected females transmit to 50% offspring; examples: vitamin D-resistant rickets, Rett syndrome (usually de novo).

Mitochondrial inheritance: maternal transmission; all children of affected female may inherit; children of affected male do not; heteroplasmy and threshold effect cause variable phenotype.

Bayesian analysis: combine prior and conditional probabilities to calculate carrier risk; example: prior 1/4, conditional (unaffected child) 1/2 → posterior (1/4 × 1/2) / [(1/4 × 1/2) + (3/4 × 1)] = 1/7.

Hardy-Weinberg equilibrium: p² + 2pq + q² = 1; carrier frequency = 2pq; if q is small, 2pq ≈ 2q; example: q = 1/50 → carrier frequency ≈ 1/25.

Relative risk (RR) and odds ratio (OR): RR = risk in exposed / risk in unexposed; OR = (a/b)/(c/d) = ad/bc; used in genetic association studies.

Penetrance adjustment: risk to offspring = 0.5 × penetrance; example: BRCA1 pathogenic variant with 60% penetrance → offspring risk = 30%.

Diagnostic testing: for symptomatic individuals; examples: cystic fibrosis (CFTR), Duchenne muscular dystrophy (DMD), Huntington disease (HTT); results guide management and reproductive decisions.

Predictive testing: for asymptomatic individuals at risk for adult-onset disorders (e.g., Huntington disease, hereditary cancer); requires pre- and post-test counseling; genetic discrimination concerns.

Carrier screening: identifies carriers of autosomal recessive and X-linked conditions; offered preconception or prenatal; examples: CF, sickle cell, Tay-Sachs, spinal muscular atrophy (SMA).

Prenatal testing: noninvasive prenatal testing (NIPT) via cell-free fetal DNA (cffDNA) from maternal blood; screening for trisomies 21, 18, 13; diagnostic tests: CVS, amniocentesis.

Newborn screening: mandated in all US states; typically includes congenital hypothyroidism, PKU, CF, sickle cell disease, hearing loss; uses dried blood spots; tandem mass spectrometry.

Pharmacogenomics: genetic variants affecting drug metabolism; examples: CYP2C19 and clopidogrel, CYP2D6 and codeine, TPMT and thiopurines, HLA-B*57:01 and abacavir.

Hereditary cancer syndromes: BRCA1/2 (breast/ovarian), Lynch syndrome (MMR genes), Li-Fraumeni (TP53), FAP (APC); surveillance and risk-reducing surgery based on gene-specific risks.

Preimplantation genetic testing (PGT): PGT-A (aneuploidy), PGT-M (monogenic), PGT-SR (structural rearrangements); requires in vitro fertilization; biopsy at blastocyst stage.

Direct-to-consumer (DTC) genetic testing: limited clinical validity; not equivalent to clinical-grade testing; results should be confirmed in CLIA-certified laboratory before clinical action.

CLIA (Clinical Laboratory Improvement Amendments): federal regulation of clinical laboratories; requires certification, proficiency testing, quality control, and personnel qualifications; enforced by CMS.

CAP (College of American Pathologists): accrediting organization; laboratory accreditation program includes inspections and proficiency testing; often deemed sufficient for CLIA compliance.

ISO 15189: international standard for medical laboratories; specifies quality and competence requirements; not a substitute for CLIA in the US.

Quality control (QC): daily monitoring of test performance; for molecular tests, include positive and negative controls; for NGS, metrics: coverage depth, uniformity, variant allele fraction, contamination.

Proficiency testing (PT): external evaluation of laboratory performance; required for CLIA-certified tests; failures require corrective action.

Validation vs verification: validation establishes performance characteristics for new tests (accuracy, precision, analytical sensitivity/specificity, reportable range, reference range); verification confirms performance of FDA-cleared tests in the laboratory's environment.

FDA regulation: in vitro diagnostic (IVD) devices; laboratory-developed tests (LDTs) currently under enforcement discretion but FDA oversight increasing; some states (e.g., New York) require additional review.

Documentation: standard operating procedures (SOPs), instrument maintenance, personnel training, competency assessment, and quality assurance records; retention per regulations (e.g., 2 years for CLIA, longer for CAP).

Biosafety: BSL-2 for most clinical molecular testing; BSL-3 for certain agents; proper waste disposal and spill procedures.

Variant nomenclature (HGVS): c. for coding DNA, p. for protein; examples: c.76A>T (p.Lys26*), c.1521_1523delCTT (p.Phe508del); use reference sequence (e.g., NM_000492.3).

Variant classification (ACMG/AMP): pathogenic (P), likely pathogenic (LP), uncertain significance (VUS), likely benign (LB), benign (B); based on evidence categories: population, computational, functional, segregation, de novo, allelic.

Population databases: gnomAD, 1000 Genomes, ESP; allele frequency thresholds: >5% likely benign; <0.01% may be pathogenic for rare Mendelian disorders; consider genetic ancestry.

In silico prediction tools: SIFT, PolyPhen-2, CADD, REVEL; used as supporting evidence (not standalone) for missense variants; splice predictors: SpliceSiteFinder, MaxEntScan.

NGS data analysis pipeline: alignment (BWA, Bowtie) → variant calling (GATK, FreeBayes) → annotation (VEP, ANNOVAR) → filtering (frequency, impact, inheritance) → interpretation.

Coverage metrics: depth (≥20x for germline, ≥500x for somatic), breadth (≥95% of target region at ≥20x), uniformity; low coverage regions require orthogonal confirmation.

Copy number variant (CNV) detection from NGS: read depth, split reads, discordant pairs; confirm with CMA or MLPA.

Sanger confirmation: required for clinically actionable variants from NGS; confirms variant and zygosity; also used to resolve ambiguous calls.

RNA analysis: for splice-site variants and deep intronic variants; can detect aberrant splicing and expression levels; complementary to DNA testing.

Genetic Information Nondiscrimination Act (GINA) of 2008: prohibits discrimination in health insurance and employment based on genetic information; does not cover life, disability, or long-term care insurance.

HIPAA Privacy Rule: protects individually identifiable health information, including genetic information; requires authorization for disclosure; applies to covered entities.

Informed consent: essential for genetic testing; must include purpose, risks, benefits, alternatives, confidentiality, and implications for family members; separate consent for research and for sensitive tests (e.g., Huntington disease).

Duty to warn (duty to disclose): obligation to inform at-risk relatives of genetic risk; varies by state; often requires patient permission; example: BRCA1 pathogenic variant.

Genetic discrimination: historical examples (e.g., sickle cell screening); GINA and state laws provide protections but gaps remain (life insurance, long-term care).

Direct-to-consumer (DTC) genetic testing: ethical concerns include privacy, data security, clinical validity, and lack of counseling; consumers should understand limitations.

Incidental/secondary findings: ACMG recommends reporting pathogenic variants in 73 genes (e.g., BRCA1/2, Lynch syndrome) unless patient opts out; requires consent.

Equity and access: genetic testing disparities by race, ethnicity, and socioeconomic status; importance of diverse reference databases and culturally competent counseling.

Return of results: policies for research and clinical settings; participants' preferences should be respected; results should be clinically actionable and confirmed.