title: Δ-PCR, A Simple Method to Detect Translocations and Insertion/Deletion Mutations.
abstract: PCR detection of chromosomal translocations and small insertion/deletion mutations is challenging when potential amplicon size varies greatly. Molecular diagnostic laboratories face such difficulties with the BCL2-IGH translocation in follicular lymphoma and with internal tandem duplication mutation of the FLT3 gene in leukemia, where breakpoints are widely distributed, mutations may be multiple, signal strength is low, and background noise is elevated. We developed a strategy, called Δ-PCR, that ensures PCR specificity and identifies individual breakpoints. Δ-PCR uses two forward primers (external and internal) and a reverse primer simultaneously. The internal primer functions as a probe with a defined distance Δ from the external primer. For follicular lymphoma, we prepared upstream, BCL2-specific primers for potential breakpoints to pair with a common, downstream VLJH primer. Multiplexed PCR amplicons are sized by capillary electrophoresis. Each of the upstream pairs has a defined interval separating them that uniquely identifies the breakpoint. The presence of two amplicons with a defined size difference confirms validity of the rearrangement and identity of the specific breakpoint, even if signal strength is low. By testing 40 follicular lymphoma and 12 control specimens from formalin-fixed, paraffin-embedded (FFPE) blocks, we showed that multiplex Δ-PCR is a simple, sensitive strategy to identify translocations with multiple breakpoints or partners. The strategy was also applied to detect minor leukemic clones with internal tandem duplication mutations and could have broader applications for other insertion/deletion and duplication mutations.
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Ming-Tseh
authors.initials: MT
authors.lastname: Lin
authors.forename: Li-Hui
authors.initials: LH
authors.lastname: Tseng
authors.forename: Roy G
authors.initials: RG
authors.lastname: Rich
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hafez
authors.forename: Shuko
authors.initials: S
authors.lastname: Harada
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Murphy
authors.forename: James R
authors.initials: JR
authors.lastname: Eshleman
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Gocke
mesh_terms: D056905:Chromosome Breakpoints; D020440:Gene Duplication; D019254:Genes, bcl-2 / Q000235:genetics; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis / Q000235:genetics; D008224:Lymphoma, Follicular / Q000175:diagnosis / Q000235:genetics; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017384:Sequence Deletion; D014178:Translocation, Genetic / Q000235:genetics*; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics
chemical_list: D007143:Immunoglobulin Heavy Chains; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3