Applications of FISH in disease

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Applications of FISH in disease

• Dr Vivi M Srivastava
• Cytogenetics Unit
• Christian Medical College, Vellore

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Fluorescence In-Situ Hybridization (FISH)

• A method used to identify the presence and
  location of a specific part of a gene or
  chromosome.
• It does not screen all the chromosomes for
  abnormalities.

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Definitions

• Genome The entire DNA of an organism
• Humans
• diploid (chromosome pairs)
• Haploid genome is one set of chromosomes - 3 x
  109 ( billion) bp per haploid genome
• Chromosome structure found within a cell nucleus
  consisting of a continuous length of ds DNA
• Humans
• 22 pairs of autosomal chromosomes
• 2 sex chromosomes

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Resolution of FISH

• Haploid human genome has
• 3 x 109 ( billion) bp
• Conventional cytogenetic analysis will detect
  abnormalities gt 5 Mb
• ( 5 x 106 bp, million) in length
• FISH detects abnormalities gt 10 -50 kb

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Definitions

• Locus a position on a chromosome
• Gene A sequence of DNA which codes for a
  specific protein that determines a particular
  characteristic or function.

                                                                                                                                                                      


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DNA strands
OH3
5p
Both strands of the DNA double helix are
complementary to each other and antiparallel
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FISH probe

• Probe
• is a length of DNA specific to one region of a
  chromosome
• is labelled with a fluorescent molecule
  throughout its length
• will attach to the complementary sequence in each
  cell / metaphase on the slide

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FISH probe
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Hybridization

• Nucleic acid hybridization - formation of a
  duplex between two complementary sequences.

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In situ Hybridization

• Test DNA immobilized on a glass slide (inert
  support) to prevent self-annealing so that they
  are available for hybridization with the
  complementary sequence of the probe.

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FISH procedure

• First step denature (unwind) the double
  stranded DNA in both the probe DNA and the test
  sample (on the slide) so they can bind to each
  other.
• Done by heating the DNA in a solution of
  formamide at a high temperature (70 -75º C)

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FISH procedure

• Next, apply probe to slide and coverslip.
• To prevent evaporation, the edges of the
  coverslip are sealed with rubber cement.
• Coverslipped slide placed in an incubator at 37º
  C overnight for the probe to hybridize with the
  target chromosome.

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FISH procedure

• During hybridisation,
• probe DNA seeks out its target sequence
• on the specific chromosome and
• binds to it.
• The strands slowly rejoin (re-anneal).

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DNA Denaturation - Renaturation
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Interphase FISH - basic steps

• Make a cell suspensionApply cells to glass
  slide.
• Denature cells and probe
• Apply probe to slide
• HybridizeWash slides to remove unbound probe
• Counterstain
• View under epifluorescence microscope

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Types of FISH Probes
Telomeric
Centromeric
Collections of small probes, each to a different
sequence along the length of the same
chromosome.
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Centromeric (Alphoid) probes

• Repetitive sequences found at the centromeres
  of chromosomes.
• Chromosome enumerator probes (CEP)- to
  determine the number of copies of a locus.

Centromeric probe for chromosome 8 showing
trisomy
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Locus specific probes

• Hybridize to a particular region of a chromosome.
  
• Used to determine
• if a gene is present in its usual position,
• the number of copies of a gene,
• on which chromosome a gene is located.

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Constitutional abnormalities
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LSI TUPLE1 (HIRA)
NORMAL 2R2G Orange signals 22q11.2 Green
signals 22q13.3
POSITIVE FOR DELETION 22q11.2 1R2G
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To detect a microdeletion syndrome FISH
confirms deletion of Prader-Willi locus
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To characterise an abnormal chromosome
Chr 10 material on chr 4q
Probe for diGeorge 2 locus on chromosome 10p
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To establish the origin of an unidentifiable
chromosome
Isochromosome 12 p in
Pallister-Killian syndrome
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Disorder of sexual differentiation
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Disorders of sexual differentiation - to
establish the presence of the SRY gene
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Disorders of sexual differentiation - to confirm
the presence of mosaicism
Monosomy X
Disomy X
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Prenatal diagnosis

• For trisomy/ sex chromosome aneuploidy/structura
  l abnormality
• Pre implantation genetic diagnosis

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To establish trisomy locus specfic probe for
chromosome 21
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FISH in Cancer
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Haematological malignancies - diagnosis and
prognosis

• Confirmation of diagnosis suspected acute
  promyelocytic leukemia, chronic myeloid leukemia
  versus other myeloproliferative disorders
• Detect cryptic abnormalities t(1221)
• Prognosis monosomy 7 in AML, t (922) in ALL

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Chronic myeloid leukemia - t(922)

Philadelphia chromosome

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t(1221) a submicroscopic abnormality
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Haematological disorders - treatment response

• Follow up of pts on treatment count number of
  cells with abnormality at diagnosis and post
  treatment.
• Engraftment status in sex-mismatched BMT

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Centromeric probes for chromosomes X and Y to
detect chimerism following sex mismatched bone
marrow transplantation
Green - Y Red -X
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Detection of abnormality when cell yield /
morphology is poor

• Confirmation of suspected abnormality if
  morphology is poor eg inv 16, t(911)
• To detect abnormality if there are not adequate
  cells for culture
• Panels for myeloma , chronic lymphocytic leukemia

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CBFß PROBE inversion 16
Poor chromosome morphology
inversion 16 1F1G1R
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Cancer solid tumours

• Gliomas 1p,19q
• Ca breast HER 2 neu amplification status
• Urine for aneuploidy
• Ca prostate
• RB1 gene in retinoblastoma
• NMYC amplification status in neuroblastoma -
  prognosis

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NMYC amplification in neuroblastoma poor
prognosis
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Localisation of a single gene (yellow dots) on a
chromosome by FISH
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Advantages and limitations of FISH

• Can be used on any tissue dividing cells not
  required
• Rapid screening of large numbers of cells
• Detects specific abnormalities
• Does not screen whole genome - will not detect
  additional abnormalities which might signify that
  there is clonal evolution
• Probes expensive

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APML with PML /RARA fusion
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APML with t(1117) negative for PML /RARA fusion
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Limitations of FISH

• Difficult to interpret small numbers of cells
  with deletions correlation with clinical and
  morphological features essential
• Cost of probe panels high
• Metaphase FISH gold standard if in doubt may
  not always be available

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Multicolour Karyotyping

• SPECTRAL KARYOTYPING (SKY) AND MULTICOLOUR FISH
  (M-FISH)
• Cell culture required
• Metaphases from a tumour hybridized with probes
  specific to each chromosome
• Each chromosome assigned a different colour by
  using four to seven different fluorochromes
• Similar principle different methods used to
  produce images

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(No Transcript)
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SKY t(1214)
SKY t(1214)
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SKY/M-FISH APPLICATIONS

• Detection of
• complex rearrangements involving three to four
  chromosomes
• marker chromosomes of unknown origin
• translocations and insertions any colour
  change upto 2-3Mb
• Novel aberrations

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LIMITATIONS OF SKY/M-FISH

• Cannot detect inversions / small gains and losses
• Probes expensive
• Reference to conventional cytogenetics
• necessary
• Specialised equipment necessary

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Array CGH

• Metaphase chromosomes replaced by cloned DNA
  arrayed on a glass slide
• Resolution can be adjusted according to need

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Summary

• FISH is a valuable test that can be used for a
  variety of diseases
• Targeted testing, so choice of correct probe is
  critical it can complement other forms of
  testing
• If used by itself, correlation with clinical
  features and morphology is essential

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Use of FISH in disease

• To determine number of copies of a locus
  trisomy/monosomy/other aneusomy
  mosaicism/amplification /other
• To establish genetic sex in disorders of sexual
  development
• Confirm/detect abnormalities translocations/delet
  ions/inversions/duplications /amplification/crypti
  c abnormalities/other

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Use of FISH in disease contd.

• To
• identify the origin of an abnormal chromosome
• establish diagnosis / prognosis
• monitor treatment response in malignancy
  including chimerism in sex mismatched bon marrow
  transplants

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Post natal

• To detect an qbnormality not seen in the kt
• To confirm a (suspected)clinical diagnosis
  pws/diG
• To confirm the sex of a patient with
  dsd/ambiguous genitalia/sex reversal/cah
• sry
• To characterise a suspected abnormality
• To detect the breakpoints
• To check if an apparently balanced t causes a
  submicroscopic deletion
• To confirm low level mosaicism

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Summary

• No single method is complete
• For routine clinical samples
• conventional cytogenetics
• FISH
• For characterization of complex karyotypes
• aCGH and / SKY or MFISH