Rearrangement versus Translocation

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					Assay for 14;18 Translocation
    PCR/Blot Hybridization


         (And what is a rearrangement?)
      Lecture Outline

   What is a translocation?
   What is the significance of a translocation?
   What is the significance of a 14;18
    translocation?
   Why is an assay for a 14;18 translocation
    useful?
   How does the assay work?
   What is a rearrangement?
What is a translocation?


Exchange of
material between
two different
chromosomes,
sometimes
reciprocal (a
trade),
sometimes,
                   Reciprocal translocation
oneway.
                       (see next slide)
 Material from Chromosome 9 is stained red.
Material from Chromosome 22 is stained green.
    Examples of translocations
   t(9;22)
       c-abl gene of Chromosome 9 juxtaposed to (placed next to)
        part of a BCR (B cell receptor/immunoglobulin) gene of C22
   t(8;14), t(2;8), t(8;22)
       c-myc gene of C8 juxtaposed to the Ig heavy- or light-chain
        locus on C14, 2, or 22
   t(11;14)
       Cyclin D1/bcl-1/PRAD-1 locus of C11 juxtaposed to an Ig
        Heavy Chain gene of C14
   t(14;18)
       bcl-2 gene of C18 juxtaposed to an Ig Heavy Chain gene of
        C14
What is the significance of
translocations?

   Some kinds of translocations go undetected
    because they have no noticeable effect.
   The translocations on the previous slides were
    detected as a result of research which showed that
    each appeared in association with certain cancers.
       Each of these translocations has been found to
        ACTIVATE a gene that causes cell division or prevents
        cell death.
            Such genes have acquired the name proto-oncogenes.
What is a proto-oncogene?
   A normal cellular gene that encodes a
    protein usually involved in regulation of
    cell proliferation and that can be
    mutated into a cancer-promoting
    oncogene, either by
       changing the protein-coding segment or by
       altering its expression.
What is the significance of
translocations? (cont’d)
   Each oncogenic translocation leads to loss of
    regulation of the activity level of a gene product
    that controls the rate of cell division or death:
       c-abl: a tyrosine kinase that stimulates cell division
       Myc: a transcription factor that activates many genes for
        cell cycle progression
       Bcl-1/PRAD-1: Cyclin D1, a protein that promotes cell
        cycle progression
       bcl-2: a gene whose expression prevents apoptotic cell
        death
   Overexpression of a gene that codes for a protein
    that stimulates cell division or that prevents cell
    death may contribute to the development of
    cancer.
             What is the significance of a
             14;18 translocation?

   The 14;18 translocation is found in the tumor cells
    of patients with a specific form of follicular B cell
    lymphoma (hence the name “bcl”).
       14;18 translocation is reported
            in 80-90% of follicular lymphomas
                 But is only one of several mutated genes in each lymphoma
                 And, 10-20% of follicular lymphomas can not be diagnosed by
                  looking for a 14;18 translocation.
            less frequently in other hematopoietic and nonhematopoietic
             malignancies
        How is detection of a 14;18
        translocation useful?

   It can support the cytological (microscopic
    technique) diagnosis of follicular lymphoma
       Helps guide physician as to prognosis and treatment
   It can be used to determine the presence of
    minimal residual disease (MRD) after treatment.
       How successful was therapy?
       Has the disease reappeared after remission?
Schematics of t(14;18)
    How does the 14/18 assay work?


   Extract DNA from patient tumor cells.
   PCR with primers flanking the translocation region.
       product expected only if translocation has occurred
   Perform agarose gel electrophoresis of PCR products.
   Blot PCR fragments from gel to hybridization
    membrane.
   Detect a translocation PCR fragment with probe for
    the sequence expected to be amplified.
   Make a diagnosis by analysis of sample and controls.
What is Bcl 2?
   Bcl 2 is a gene whose product is involved in
    blocking apoptosis.
   Bcl 2 is normally expressed in long-lived B
    and T cells
   After translocation to chromosome 14, Bcl 2
    is overexpressed as a result of deregulation
    of expression and/or as a result of the activity
    of the enhancer region of the immunoglobulin
    heavy chain.
Bcl 2 continued
   Bcl 2 expression is important in cells such as
    T and B cells that use apoptosis as a
    prominant control mechanism.
   Apoptosis may be important in inhibiting the
    growth of potentially tumorigenic cells.
   When apoptosis is prevented by Bcl 2 activity,
    the cells survive instead of dying.
What primers are used to identify the
14:18 translocation?
   One set of primers is used to identify
    the MBR/JH translocation and another
    set is used to identify the MCR/JH
    translocation.
   Note that you are doing only half of this
    test - the half that can identify a
    MBR/JH translocation.
How are primers and probe chosen?

            
                  --------------




  One primer for Chromosome 18.
  Complementary 5’ of breakpoint.
  No PCR occurs without a translocation.
          (mbr = major breakpoint REGION)
          (mcr = minor breakpoint cluster REGION)
How are primers and probe chosen?

 One primer for Chromosome 14.
 Complementary 3’ of breakpoint.
 No PCR occurs without a translocation.




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     
But at a translocation . . .
             





              Successful PCR
            ———
            ———
            ———
            ———
            ———
            ———
            ———
            ———
Probe

  ———
  ———
   _
         MBI oligonucleotide probe
         complementary to
        sequence interior to primers
How is PCR used to identify a
chromosomal translocation?
   Thus, a product will be generated only
    if a translocation brings together, on a
    single chromosome, the regions to
    which both of the primers bind.
What size product will be generated
using the two different sets of
primers?
   The size of the product will vary
    depending upon the exact locations of
    each of the breakpoints in the two
    chromosomes involved.
   For our assay using the MBR/JH primer
    pair, the product that we should get
    from the positive cell line that we used
    is ~235 basepairs.
Blot hybridization of PCR products from several different
  follicular B cell lymphoma samples with MBR probe
Essential components

   Sample - DNA extracted from tumor tissue
   Detection scheme - PCR followed by
    Southern blotting and hybridization
       Specificity
            PCR primers
            Oligonucleotide probe complementary to PCR product
       Visualization
            Oligonucleotide probe conjugated to the enzyme
             alkaline phosphatase
            Alkaline-phosphatase action on CDP-Star substrate 
             chemiluminescence recorded on film
    Essential components
   Sensitivity
       Amplification of chromosomal region of interest
        by PCR
       Amplification of signal for positive hybridization
        by repetitive alkaline phosphatase activity
       Reduction of background noise by carefully
        chosen conditions for
            PCR
            Hybridization
            Washes following hybridization and during
             visualization
    Essential components
Controls
As you perform the assay, look for how the controls
  below are incorporated.

   Controls for PCR
       Correct primers are being used for the translocation of
        interest
       Contaminant DNA is not present in PCR reagents
       Patient DNA is amplifiable
            Hint: one set of primers amplifies part of the gene for beta-globin,
             which is a hemoglobin gene irrelevant to lymphoma, located on
             Chromosome 11, and normal in “everyone”.
Essential components

Controls
As you perform the assay, look for how the
  controls below are incorporated.

   Controls for hybridization and visualization
       Stringency conditions appear to be appropriate
       Visualization system is working
So What Is a Rearrangement?
   Rearrangement is a term used to describe a
    recombination of DNA within a single
    chromosome.
Rearrangement
   Remember that every immunoglobulin
    molecule and every TCR contains both a
    variable region and a constant region and
    that the variable region is the part of the
    molecule that interacts with the antigen.
TCR = T cell receptor
Immunoglobulin = Antibody = B cell receptor (BCR)
What is meant by the term
rearrangement in relation to DNA?
   Antigen independent maturation of B and T
    cells to immunocompetent B and T cells that
    express receptors for antigen recognition
    involves a rearrangement of the cell’s
    germline DNA.
       Germline DNA = DNA that is an exact replicate of
        the genome present at fertilization of egg by
        sperm. It has not been rearranged.
Rearrangement continued
   The genes that code for the different chains
    of the B and T cell receptors include a large
    number of exons.
   The exons that code for the variable regions
    of the immunoglobulin molecules and TCRs
    are called the variable (V) segments, diversity
    (D) segments, and junctional (J) segments,
    and those that code for the constant regions
    are called the C segments.
Shematic of
germline genes
for TCR alpha
and beta and
immunoglobulin
heavy and kappa
light chains
Rearrangement continued
   For the immunoglobulin heavy chain,
    there are 100-200 different V regions,
    30 different D regions, 6 different J
    regions, and 9 different C regions.
   For the TCR beta chain, there are 75-
    100 different V regions, 2 different D
    regions, 13 different J regions, and 2
    different C regions.
Rearrangement continued
   The first step in the rearrangement process
    involves the the selective apposition of one D
    segment of the gene with one J segment by
    deletion of all intervening sequences, both
    coding and non-coding (a DJ rearrangement).
   In a similar process a V segment becomes
    apposed to the DJ rearrangement to form a
    VDJ rearrangement.
Rearrangement continued
   After rearrangement of the DNA,
    transcription occurs.
   Subsequent splicing of the mRNA with
    deletion of noncoding sequences results
    in apposition of VDJ to C to form a
    VDJC mRNA which can be translated
    into an immunoglobulin heavy chain or
    TCR.
Rearrangement continued
   Once the productive rearrangement occurs on
    one chromosome, an active protein chain is
    synthesized and it provides a trigger to
    prevent rearrangement of the other allele
    (allelic exclusion).
   Thus each B or T cell expresses only one
    immunoglobulin or TCR, respectively.
Rearrangement continued
   This complex process of rearrangement
    allows for tremendous diversity of both
    the cell mediated and humoral arms of
    the immune system, and the ability to
    respond to a wide array of antigens.
Review: How do translocation and
rearrangement differ?
   Translocation is the recombination of
    DNA between two different
    chromosomes. Rearrangement is the
    recombination of DNA within a single
    chromosome.
   Translocations are abnormal.
    Rearrangements of genes for
    immunoglobulins and TCRs are normal.

				
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posted:6/12/2012
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