Chromosomal Structure and Chromosomal Mutations Chapter 8 Chromosomal Structure and Chromosomal Mutations

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.

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

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

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

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

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

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: 30nm Metaphase (Mitosis) Extended metaphase chromosomes: 300 nm Condensed metaphase chromosomes: 700 nm

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

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

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

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

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

Normal Female Karyotype (High-Resolution G Banding)

Chromosome Number Abnormality Aneuploidy (48, XXXX)

Chromosome Number Abnormality Trisomy 21 (47, XX, +21)

Chromosome Structure Abnormalities Translocation Deletion Insertion Inversion Isochromosome Ring chromosome Derivative

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

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)

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

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).

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

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

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

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

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

Translocation by Metaphase FISH WCP Probe (Whole-Chromosome Painting)

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.