4/8/2013
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Chromosomal Structure and Chromosomal Mutations Chapter 8
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
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. Copyright © 2012 F.A. Davis Company
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4/8/2013
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
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
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Types of Mutations Genomic: abnormal chromosome number (monosomy, polysomy, aneuploidy) Chromosomal: abnormal chromosome structure Gene: DNA sequence changes in specific genes Copyright © 2012 F.A. Davis Company
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
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 Copyright © 2012 F.A. Davis Company
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Chromosome Morphology Telomere
Short arm (p)
Centromere
Arm
Long arm (q) Telomere
Metacentric
Submetacentric
Acrocentric
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Defining Chromosomal Location Arm
Region Band Subband 2
p 1
1
2 1 1
1 2
1 2
q 3 2 Chromosome 17
3 2 1 2 1 5 4 3 2 1
4
17q11.2
3 1 2 3 1 2, 3 4 1 2 3
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Chromosome Morphology Changes During the Cell Division Cycle DNA double helix: 2 nm diameter Interphase (G1, S, G2) Chromatin “beads on a string”: 11 nm Chromatin in nucleosomes: 30 nm Metaphase (mitosis) Extended metaphase chromosomes: 300 nm Condensed metaphase chromosomes: 700 nm Copyright © 2012 F.A. Davis Company
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Cell Division Cycle
G1
Interphase (11–30 nm fibers)
S G2 M Mitosis: Prophase Anaphase Metaphase Telophase
Metaphase (300–700 nm fibers)
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
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 Copyright © 2012 F.A. Davis Company
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Visualizing Metaphase Chromosomes (Banding) Giemsa‐, reverse‐, or centromere‐stained metaphase chromosomes
G-bands
R-bands
C-bands
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Karyotype International System for Human Cytogenetic Nomenclature (ISCN) 46, XX: normal female 46, XY: normal male
G‐banded chromosomes are identified by band pattern. Copyright © 2012 F.A. Davis Company
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Normal Female Karyotype (46, XX) (G‐banding)
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Normal Female Karyotype (High‐Resolution G‐banding)
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Chromosome Number Abnormality Aneuploidy (48, XXXX)
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Chromosome Number Abnormality Trisomy 21 (47, XX, +21)
Copyright © 2012 F.A. Davis Company
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Chromosome Structure Abnormalities
Translocation
Deletion
Derivative chromosome
Inversion
Insertion
Isochromosome
Ring chromosome
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Chromosome Structure Abnormality: Balanced Translocation 45, XY, t(14q21q)
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
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) Copyright © 2012 F.A. Davis Company
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Fluorescent in situ Hybridization (FISH) Metaphase FISH Chromosome painting Spectral karyotyping
Interphase FISH
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Uses of Fluorescent in situ Hybridization (FISH) Identification and characterization of numerical and structural chromosome abnormalities Detection of microscopically invisible deletions Detection of subtelomeric aberrations Prenatal diagnosis of the common aneuploidies (interphase FISH)
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
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
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
FISH Probes Telomere‐specific probes (TEL) Hybridize to subtelomeric regions Detect subtelomeric deletions and rearrangements
Probe binding site 100–200 kb Telomere associated repeats
Unique sequences
Telomere 3–20 kb (TTAGGG)n
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Genetic Abnormalities by Interphase FISH LSI Probe Greater or fewer than two signals per nucleus is Cell nucleus considered abnormal. Normal diploid signal
Trisomy or insertion
Monosomy or deletion Copyright © 2012 F.A. Davis Company
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Structural Abnormality by Interphase FISH LSI Probe (Fusion Probe)
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Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Structural Abnormality by Interphase FISH LSI Probe (Break Apart Probe)
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4/8/2013
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
Translocation by Metaphase FISH WCP Probe (Whole‐Chromosome Painting)
Copyright © 2012 F.A. Davis Company
Molecular Diagnostics
Fundamentals, Methods and Clinical Applications
Second Edition
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. Copyright © 2012 F.A. Davis Company
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