Chapter 8Chapter 8

Chromosomal Structure and Chromosomal Mutations

ObjectivesObjectives

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.

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

Mutations and PolymorphismsMutations and Polymorphisms

Types of MutationsTypes of Mutations

Genomic: abnormal chromosome number (monosomy, polysomy, aneuploidy)

Chromosomal: abnormal chromosome structure

Gene: DNA sequence changes in specific genes

Chromosome MorphologyChromosome 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

Centromere

Telomere

Metacentric Submetacentric Acrocentric

Telomere

Arm

Longarm (q)

Shortarm (p)

Chromosome MorphologyChromosome Morphology

p

q

Arm Region Band Subband

2

1

1

2

21

1

12

3

4

3212154321

12

312312, 34123

17q11.2

Chromosome 17

Defining Chromosomal LocationDefining Chromosomal Location

Chromosome Morphology Changes Chromosome Morphology Changes During the During the Cell Division Cycle.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 CycleCell Division Cycle

G1 S

G2

Interphase(11–30 nm fibers)

Metaphase(300–700 nm fibers)

MMitosis:ProphaseAnaphaseMetaphaseTelophase

Visualizing Metaphase Visualizing Metaphase ChromosomesChromosomes

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 Visualizing Metaphase Chromosomes (Chromosomes (BandingBanding))

Giemsa-, reverse- or centromere-stained metaphase chromosomes

G-Bands R-Bands C-Bands

KaryotypeKaryotype

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

Normal Female KaryotypeNormal Female Karyotype(High-Resolution G Banding)(High-Resolution G Banding)

Chromosome Number AbnormalityChromosome Number AbnormalityAneuploidy (48, XXXX)Aneuploidy (48, XXXX)

Chromosome Number AbnormalityChromosome Number AbnormalityTrisomy 21 (47, XX, +21)Trisomy 21 (47, XX, +21)

Translocation Deletion

Insertion

Inversion Isochromosome

Ringchromosome

Derivativechromosome

Chromosome Structure Chromosome Structure AbnormalitiesAbnormalities

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

Probe

Interphase or metaphasecells on slide (in situ)

Microscopicsignal (interphase)

Fluorescent Fluorescent in situin situ Hybridization Hybridization (FISH)(FISH)

Hybridization of complementary gene- or region-specific fluorescent probes to chromosomes.

Fluorescent Fluorescent in situin situ Hybridization Hybridization (FISH) (FISH)

Metaphase FISH Chromosome painting Spectral karyotyping

Interphase FISH

Uses of Fluorescent Uses of Fluorescent in situin situ Hybridization (FISH)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 ProbesFISH 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

Telomere

(TTAGGG)n

100–200 kb 3–20 kbUnique sequences Telomere associated repeats

Probe binding site

FISH ProbesFISH Probes

Telomere-specific probes (TEL) Hybridize to subtelomeric regions Detect subtelomeric deletions and

rearrangements

Normal diploid signal

Trisomy or insertion

Monosomy or deletion

Cellnucleus

Genetic Abnormalities by Genetic Abnormalities by Interphase FISH LSIInterphase FISH LSI Probe Probe

Greater or less than two signals per nucleus is considered abnormal.

Structural Abnormality by Interphase Structural Abnormality by Interphase FISH LSIFISH LSI Probe ( Probe (Fusion ProbeFusion Probe))

Structural Abnormality by Interphase Structural Abnormality by Interphase FISH LSIFISH LSI Probe ( Probe (Break Apart ProbeBreak Apart Probe))

Translocation by Metaphase FISHTranslocation by Metaphase FISHWCP Probe (WCP Probe (Whole-Chromosome PaintingWhole-Chromosome Painting))

SummarySummary

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.