Principles of Molecular Pathology

Principles of Molecular Pathology - Gene & Genome Basics

• DNA vs. RNA Structure:
  • DNA: Deoxyribose sugar, Thymine base, Double helix.
  • RNA: Ribose sugar, Uracil base, Single strand.
• Gene: Basic hereditary unit; a DNA segment coding for a functional product (RNA/protein).
• Genome: An organism’s complete set of DNA.
• Chromosome: Structure within cells containing genetic material (DNA).
• Central Dogma of Molecular Biology:

• Mutations: Permanent alterations in DNA sequence.
  • Point Mutation: Change in a single nucleotide.
    • Silent: No change in amino acid.
    • Missense: Results in a different amino acid.
    • Nonsense: Creates a premature stop codon.
  • Frameshift Mutation: Insertion or deletion of nucleotides (not in multiples of three); alters the reading frame.
• Polymorphism: Common variation in DNA sequence within a population.

  ⭐ Single Nucleotide Polymorphisms (SNPs) are the most common type of genetic variation in the human genome.

• Allele: Variant form of a given gene.
• Genotype: An individual's collection of genes.
• Phenotype: Observable characteristics resulting from genotype-environment interaction.

Molecular Techniques - Lab Sleuth Kit

• Polymerase Chain Reaction (PCR): Amplifies specific DNA segments.
  • Principle: Exponential DNA amplification.
  • Steps: Denaturation, Annealing, Extension.

-   Types: RT-PCR (RNA template), qPCR (quantitative real-time).

• Blotting Techniques: 📌 SNoW DRoP mnemonic:

  • Southern: DNA detection.
  • Northern: RNA detection.
  • Western: Protein detection.

• Hybridization: Probes (labeled DNA/RNA) bind to target sequences.

  • In Situ Hybridization (ISH): Detects sequences in cells/tissues.
  • Fluorescent ISH (FISH): Visualizes genes; detects gene amplification (e.g., HER2 in breast cancer), translocations (e.g., BCR-ABL in CML).

• Microarrays: Analyze expression of thousands of genes simultaneously.

• Sequencing:

  • Sanger Sequencing: Dideoxy chain termination method for determining nucleotide sequence.
  • Next-Generation Sequencing (NGS): High-throughput, parallel sequencing; faster & cost-effective for large genomes/panels.

⭐ Polymerase Chain Reaction (PCR) can amplify a single copy of DNA by millions of times, enabling detection of minute quantities of genetic material.

Clinical Applications - Disease Detectives

Molecular pathology identifies disease mechanisms:

• Genetic Disorders:
  • Monogenic: Detects CFTR mutations (Cystic Fibrosis), HBB gene variants (Sickle Cell Anemia).
  • Chromosomal: Identifies Trisomy 21 (Down Syndrome).
  • Trinucleotide Repeats: Diagnoses Huntington's Disease (expanded HTT gene repeats).
• Cancer:
  • Oncogenes (RAS, MYC) & Tumor Suppressors (TP53, RB1) for diagnosis/prognosis.
  • Therapeutic Markers: EGFR mutations (lung cancer), HER2 amplification (breast cancer).
  • Fusion Genes: BCR-ABL (Chronic Myeloid Leukemia - CML).
• Infectious Diseases:
  • Pathogen ID & Load: HIV viral load monitoring, HPV typing for cancer risk.
  • Antimicrobial Resistance: Detects genes like mecA (MRSA), NDM-1.

⭐ The Philadelphia chromosome, resulting from the t(9;22) translocation creating the BCR-ABL fusion gene, is a hallmark of Chronic Myeloid Leukemia (CML).

Emerging Concepts - Pathology's Future

• Epigenetics: Heritable changes not altering DNA sequence.
  • DNA Methylation: Gene silencing; aberrant in cancer.
  • Histone Modification: Alters chromatin accessibility.
• Pharmacogenomics: Tailoring drug therapy to genetic makeup.
  • E.g., CYP enzyme variants (warfarin sensitivity).

  ⭐ Pharmacogenomics aims to tailor drug therapy based on an individual's genetic makeup, improving efficacy and reducing adverse drug reactions.

• Liquid Biopsy: Non-invasive tumor monitoring via circulating tumor DNA (ctDNA).
  • Applications: Early detection, monitoring response, resistance.
• Gene Therapy: Introducing genetic material to treat disease.
  • Viral vectors common for delivery.
• CRISPR-Cas9: Genome editing tool for precise DNA modification (brief mention).

High‑Yield Points - ⚡ Biggest Takeaways

• Central Dogma (DNA → RNA → Protein) underpins all molecular processes.
• PCR enables DNA amplification for diagnostics; RT-PCR for RNA.
• Blotting: Southern (DNA), Northern (RNA), Western (Protein) detect specific macromolecules.
• FISH visualizes gene loci and chromosomal aberrations directly in cells.
• NGS provides high-throughput DNA/RNA sequencing for comprehensive genomic analysis.
• Monoclonal antibodies offer specific targeting in diagnostics and therapeutics.
• Epigenetics (e.g., DNA methylation) regulates gene expression without altering DNA sequence.