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Chromosomal Structure and Mutations

This chapter explores mutations and polymorphisms, distinguishing between genomic, chromosomal, and gene mutations. It discusses chromosome morphology, karyotyping, and techniques such as FISH for visualizing metaphase chromosomes. The chapter also covers chromosomal abnormalities and their detection using FISH probes.

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Chromosomal Structure and Mutations

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  1. Chapter 8 Chromosomal Structure and Chromosomal Mutations

  2. Objectives • Define mutations and polymorphisms. • Distinguish the three types of DNA mutations: genome, chromosomal, and gene. • Diagram a human chromosome and label the centromere, q arm, p arm, and telomere. • Illustrate the different types of structural mutations that occur in chromosomes. • Show how karyotypes reveal chromosomal abnormalities. • Describe interphase and metaphase FISH analyses.

  3. Mutations and Polymorphisms • Mutation: a permanent transmissable change in the genetic material, usually in a single gene • Polymorphism:two or more genetically determined, proportionally represented phenotypes in the same population

  4. Types of Mutations • Genomic: abnormal chromosome number (monosomy, polysomy, aneuploidy) • Chromosomal: abnormal chromosome structure • Gene: DNA sequence changes in specific genes

  5. Chromosome Morphology • Telomere: chromosome ends • Centromere: site of spindle attachment • Constriction of the metaphase chromosome at the centromere defines two arms • Nucleosome: DNA double helix wrapped around histone proteins

  6. Chromosome Morphology Telomere Short arm (p) Arm Centromere Long arm (q) Telomere Metacentric Submetacentric Acrocentric

  7. Arm Region Band Subband 3 2 2 1 2 2 1 p 1 5 1 4 1 3 2 1 1 17 q 1 1 . 2 2 1 1 3 1 2 2 q 3 1 3 2, 3 4 1 2 2 4 Chromosome 17 3 Defining Chromosomal Location

  8. Chromosome Morphology Changes During the Cell Division Cycle. • DNA double helix: 2nm diameter Interphase (G1, S, G2) • Chromatin “beads on a string:” 11nm • Chromatin in nucleosomes: 30nmMetaphase (Mitosis) • Extended metaphase chromosomes: 300 nm • Condensed metaphase chromosomes: 700 nm

  9. Cell Division Cycle Metaphase (300–700 nm fibers) Interphase (11–30 nm fibers) G1 S G2 M Mitosis: Prophase Anaphase Metaphase Telophase

  10. Visualizing Metaphase Chromosomes • Patient cells are incubated and divide in tissue culture. • Phytohemagglutinin (PHA): stimulates cell division • Colcemid: arrests cells in metaphase • 3:1 Methanol:Acetic Acid: fixes metaphase chromosomes for staining

  11. Giemsa-, reverse- or centromere-stained metaphase chromosomes Visualizing Metaphase Chromosomes (Banding) G-Bands R-Bands C-Bands

  12. Karyotype • International System for Human Cytogenetic Nomenclature (ISCN) • 46, XX – normal female • 46, XY – normal male • G-banded chromosomes are identified by band pattern.

  13. Normal Female Karyotype (46, XX)(G Banding)

  14. Normal Female Karyotype(High-Resolution G Banding)

  15. Chromosome Number AbnormalityAneuploidy (48, XXXX)

  16. Chromosome Number AbnormalityTrisomy 21 (47, XX, +21)

  17. Deletion Translocation Inversion Isochromosome Insertion Ring chromosome Derivative chromosome Chromosome Structure Abnormalities

  18. Chromosome Structure Abnormality:Balanced Translocation 45, XY, t(14q21q)

  19. Fluorescent in situ Hybridization (FISH) • Hybridization of complementary gene- or region-specific fluorescent probes to chromosomes. Interphase or metaphase cells on slide (in situ) Probe Microscopic signal (interphase)

  20. Fluorescent in situ Hybridization (FISH) • Metaphase FISH • Chromosome painting • Spectral karyotyping • Interphase FISH

  21. Uses of Fluorescent in situ Hybridization (FISH) • Identification and characterization of numerical and structural chromosome abnormalities. • Detection of microscopically invisible deletions. • Detection of sub-telomeric aberrations. • Prenatal diagnosis of the common aneuploidies (interphase FISH).

  22. FISH Probes • Chromosome-specific centromere probes (CEP) • Hybridize to centromere region • Detect aneuploidy in interphase and metaphase • Chromosome painting probes (WCP) • Hybridize to whole chromosomes or regions • Characterize chromosomal structural changes in metaphase cells • Unique DNA sequence probes (LSI) • Hybridize to unique DNA sequences • Detect gene rearrangements, deletions, and amplifications

  23. FISH Probes • Telomere-specific probes (TEL) • Hybridize to subtelomeric regions • Detect subtelomeric deletions and rearrangements Probe binding site Telomere 100–200 kb 3–20 kb Unique sequences Telomere associated repeats (TTAGGG)n

  24. Genetic Abnormalities by Interphase FISH LSI Probe • Greater or less than two signals per nucleus is considered abnormal. Cell nucleus Normal diploid signal Trisomy or insertion Monosomy or deletion

  25. Structural Abnormality by Interphase FISH LSI Probe (Fusion Probe)

  26. Structural Abnormality by Interphase FISH LSI Probe (Break Apart Probe)

  27. Translocation by Metaphase FISHWCP Probe (Whole-Chromosome Painting)

  28. Summary • Mutations are heritable changes in DNA. • Mutations include changes in chromosome number, structure, and gene mutations. • Chromosomes are analyzed by Giemsa staining and karyotyping. • Karyotyping detects changes in chromosome number and large structural changes. • Structural changes include translocation, duplication, and deletion of chromosomal regions. • More subtle chromosomal changes can be detected by metaphase or interphase FISH.

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