title: Genomic inverse PCR for exploration of ligated breakpoints (GIPFEL), a new method to detect translocations in leukemia.
abstract: Here we present a novel method "Genomic inverse PCR for exploration of ligated breakpoints" (GIPFEL) that allows the sensitive detection of recurrent chromosomal translocations. This technique utilizes limited amounts of DNA as starting material and relies on PCR based quantification of unique DNA sequences that are created by circular ligation of restricted genomic DNA from translocation bearing cells. Because the complete potential breakpoint region is interrogated, a prior knowledge of the individual, specific interchromosomal fusion site is not required. We validated GIPFEL for the five most common gene fusions associated with childhood leukemia (MLL-AF4, MLL-AF9, MLL-ENL, ETV6-RUNX1, and TCF3-PBX1). A workflow of restriction digest, purification, ligation, removal of linear fragments and precipitation enriching for circular DNA was developed. GIPFEL allowed detection of translocation specific signature sequences down to a 10-4 dilution which is close to the theoretical limit. In a blinded proof-of-principle study utilizing DNA from cell lines and 144 children with B-precursor-ALL associated translocations this method was 100% specific with no false positive results. Sensitivity was 83%, 65%, and 24% for t(4;11), t(9;11) and t(11;19) respectively. Translocation t(12;21) was correctly detected in 64% and t(1;19) in 39% of the cases. In contrast to other methods, the characteristics of GIPFEL make it particularly attractive for prospective studies.
authors.affiliation: Department of Genetics, Friedrich Alexander University, Erlangen, Germany.
authors.forename: Elisa
authors.initials: E
authors.lastname: Fueller
authors.affiliation: Department of Pediatric Oncology, Hematology and Clinical Immunology, University Children's Hospital, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Schaefer
authors.affiliation: Department of Pediatric Oncology, Hematology and Clinical Immunology, University Children's Hospital, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Fischer
authors.affiliation: Department of Pediatric Oncology, Hematology and Clinical Immunology, University Children's Hospital, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany.
authors.forename: Pina F I
authors.initials: PF
authors.lastname: Krell
authors.affiliation: Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Stanulla
authors.affiliation: Department of Pediatric Oncology, Hematology and Clinical Immunology, University Children's Hospital, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany.
authors.forename: Arndt
authors.initials: A
authors.lastname: Borkhardt
authors.affiliation: Department of Genetics, Friedrich Alexander University, Erlangen, Germany.
authors.forename: Robert K
authors.initials: RK
authors.lastname: Slany
mesh_terms: D002648:Child; D056905:Chromosome Breakpoints; D002880:Chromosomes, Human, Pair 11; D002881:Chromosomes, Human, Pair 12; D002888:Chromosomes, Human, Pair 19; D002891:Chromosomes, Human, Pair 21; D002894:Chromosomes, Human, Pair 4; D002899:Chromosomes, Human, Pair 9; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D051788:Myeloid-Lymphoid Leukemia Protein / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis / Q000235:genetics*; D012680:Sensitivity and Specificity; D014178:Translocation, Genetic; D000097764:ETS Translocation Variant 6 Protein
chemical_list: D050676:Core Binding Factor Alpha 2 Subunit; D004270:DNA, Circular; D051788:Myeloid-Lymphoid Leukemia Protein; D015514:Oncogene Proteins, Fusion; C113855:MLL-AF4 fusion protein, human; C474558:MLL-AF9 fusion protein, human; C425207:MLL-ENL oncoprotein, human; C543638:TCF3-PBX1 fusion protein, human; C102421:TEL-AML1 fusion protein; D000097764:ETS Translocation Variant 6 Protein