VIEWS: 3 PAGES: 42 POSTED ON: 6/12/2012
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. -------- 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.
Pages to are hidden for
"Rearrangement versus Translocation"Please download to view full document