Genomic inverse PCR for exploration of ligated breakpoints (GIPFEL), a new method to detect translocations in leukemia

PLoS One. 2014 Aug 19;9(8):e104419. doi: 10.1371/journal.pone.0104419. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Child
  • Chromosome Breakpoints*
  • Chromosomes, Human, Pair 11
  • Chromosomes, Human, Pair 12
  • Chromosomes, Human, Pair 19
  • Chromosomes, Human, Pair 21
  • Chromosomes, Human, Pair 4
  • Chromosomes, Human, Pair 9
  • Core Binding Factor Alpha 2 Subunit / genetics
  • DNA, Circular / chemistry
  • DNA, Circular / genetics*
  • Humans
  • Myeloid-Lymphoid Leukemia Protein / genetics
  • Oncogene Proteins, Fusion / genetics
  • Polymerase Chain Reaction / methods*
  • Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / diagnosis
  • Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / genetics*
  • Sensitivity and Specificity
  • Translocation, Genetic*

Substances

  • Core Binding Factor Alpha 2 Subunit
  • DNA, Circular
  • MLL-AF4 fusion protein, human
  • MLL-AF9 fusion protein, human
  • MLL-ENL oncoprotein, human
  • Oncogene Proteins, Fusion
  • TCF3-PBX1 fusion protein, human
  • TEL-AML1 fusion protein
  • Myeloid-Lymphoid Leukemia Protein

Grants and funding

This research was funded by a grant of the German Ministry for Radiation Protection (Bundesministerium für Strahlenschutz, BfS) grant number 3612S70019. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.