Chromosomal Structure and Chromosomal Mutations

4/8/2013 Molecular Diagnostics Fundamentals, Methods and Clinical Applications Second Edition Chromosomal Structure and  Chromosomal Mutations Cha...
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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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4/8/2013

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

Copyright © 2012 F.A. Davis Company

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4/8/2013

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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4/8/2013

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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4/8/2013

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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4/8/2013

Molecular Diagnostics

Fundamentals, Methods and Clinical Applications

Second Edition

Normal Female Karyotype (46, XX) (G‐banding)

Copyright © 2012 F.A. Davis Company

Molecular Diagnostics

Fundamentals, Methods and Clinical Applications

Second Edition

Normal Female Karyotype (High‐Resolution G‐banding)

Copyright © 2012 F.A. Davis Company

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4/8/2013

Molecular Diagnostics

Fundamentals, Methods and Clinical Applications

Second Edition

Chromosome Number Abnormality Aneuploidy (48, XXXX)

Copyright © 2012 F.A. Davis Company

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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4/8/2013

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)

Copyright © 2012 F.A. Davis Company

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4/8/2013

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

Copyright © 2012 F.A. Davis Company

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4/8/2013

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

Copyright © 2012 F.A. Davis Company

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4/8/2013

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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4/8/2013

Molecular Diagnostics

Fundamentals, Methods and Clinical Applications

Second Edition

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

Copyright © 2012 F.A. Davis Company

Molecular Diagnostics

Fundamentals, Methods and Clinical Applications

Second Edition

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

Copyright © 2012 F.A. Davis Company

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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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