Genomic Inverse PCR for Exploration of Ligated Breakpoints (GIPFEL), a New Method to Detect Translocations in Leukemia

<div><p>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.</p></div

A New Workflow for Whole-Genome Sequencing of Single Human Cells

Binder V, Bartenhagen C, Okpanyi V, et al. A New Workflow for Whole-Genome Sequencing of Single Human Cells. Human mutation. 2014;35(10):1260-1270.: Unbiased amplification of the whole-genome amplification (WGA) of single cells is crucial to study cancer evolution and genetic heterogeneity, but is challenging due to the high complexity of the human genome. Here, we present a new workflow combining an efficient adapter-linker PCR-based WGA method with second-generation sequencing. This approach allows comparison of single cells at base pair resolution. Amplification recovered up to 74% of the human genome. Copy-number variants and loss of heterozygosity detected in single cell genomes showed concordance of up to 99% to pooled genomic DNA. Allele frequencies of mutations could be determined accurately due to an allele dropout rate of only 2%, clearly demonstrating the low bias of our PCR-based WGA approach. Sequencing with paired-end reads allowed genome-wide analysis of structural variants. By direct comparison to other WGA methods, we further endorse its suitability to analyze genetic heterogeneity

Flow chart of the GIPFEL procedure.

<p>A. Biochemical steps for enrichment of circularized DNA. The products of a restriction enzyme (E) digest of genomic material are column purified and ligated in a large volume. Subsequently exonuclease III (presented in yellow) removes remaining linear fragments allowing enrichment for circularized DNA. B. PCR strategy to detect the presence of translocation specific circles. Primer pairs are designed that cover all possible ligation joints of translocation specific ligation products. Semi-nested PCR is performed first with an outer primer corresponding to the 5′ portion of the fusion and pools of downstream primers. The PCR products from these reactions are used as templates for secondary PCRs using a 5′ inner primer and the same downstream primers, yet in different combinations. A control PCR amplifies a ligation joint created from wild-type cells. C. Decision tree for scoring of GIPFEL results.</p

Next-generation-sequencing-spectratyping reveals public T-cell receptor repertoires in pediatric very severe aplastic anemia and identifies a beta chain CDR3 sequence associated with hepatitis-induced pathogenesis

Krell P, Reuther S, Fischer U, et al. Next-generation-sequencing-spectratyping reveals public T-cell receptor repertoires in pediatric very severe aplastic anemia and identifies a beta chain CDR3 sequence associated with hepatitis-induced pathogenesis. Haematologica. 2013;98(9):1388-1396

Examples of GIPFEL results.

<p>A. Sensitivity test. Circularized genomic DNA was produced from MV4;11 cells a cell line with a known t(4;11) translocation and from HL60 cells as “non-translocation” control as well as from various mixtures “diluting” MV4;11 cells in a population of HL60 as indicated. GIPFEL was performed and real-time amplification curves are shown. B. As in “A” with REH t(12;21) cells and 697 cells instead of HL60 cells. C. As in “A” with 697 t(1;19) and REH cells. D. Example for a GIPFEL result using patient DNA. Upper panel: Amplification chart of a typical GIPFEL experiment with patient DNA. Amplification is achieved with the genomic MLL control primer and a translocation specific primer pair. Lower panel: Agarose gel electrophoresis of the 8 individual secondary PCRs interrogating the (4;11) breakpoint region. E. Results presented as in “D” for a t(12;21) breakpoint. F. Results for a t(1;19) patient sample.</p

Primers used for GIPFEL.

<p>Primers used for GIPFEL.</p

Breakpoint distribution, restriction site and primer locations for individual translocations.

<p>A. Schematic depiction of the 11q23 breakpoint region covered by GIPFEL. Consecutively numbered BamHI sites (B), primer locations (arrows) and exons (squares) involved are depicted. Numbers denote the size in kb between restriction sites. * Note: For restriction fragments <1 kb no primers were designed. B. Schematic depiction of the t(12;21) breakpoint regions covered by GIPFEL. SacI sites (S), primer locations and exons involved are depicted as described in A. Numbers denote the size in kb between restriction sites. † Note: Restriction sites S9 and S10 were 4 bp apart. No primer was designed for site S9. C. Schematic depiction of the t(1;19) breakpoint covered by GIPFEL. Presentation as in A and B. Digest was carried out with MfeI (M). The heatmap indicates the frequency of the breakpoints detected in the respective region.</p

GIPFEL results summary.

<p>* =  two different breakpoints were detected in a patient sample.</p

Basic principle of GIPFEL.

<p>Upon restriction digest and circularization of genomic DNA only genomic DNA from translocation bearing cells will form circles that join DNA of two different chromosomes. The junction is predetermined by the location of the genomic breakpoint. By probing for all possible ligation junctions with PCR the presence of a translocation can be ascertained.</p

Next-generation-sequencing of recurrent childhood high hyperdiploid acute lymphoblastic leukemia reveals mutations typically associated with high risk patients

Chen C, Bartenhagen C, Gombert M, et al. Next-generation-sequencing of recurrent childhood high hyperdiploid acute lymphoblastic leukemia reveals mutations typically associated with high risk patients. Leukemia research. 2015;39(9):990-1001.: 20% of children suffering from high hyperdiploid acute lymphoblastic leukemia develop recurrent disease. The molecular mechanisms are largely unknown. Here, we analyzed the genetic landscape of five patients at relapse, who developed recurrent disease without prior high-risk indication using whole-exome- and whole-genome-sequencing. Oncogenic mutations of RAS pathway genes (NRAS, KRAS, FLT3, n=4) and deactivating mutations of major epigenetic regulators (CREBBP, EP300, each n=2 and ARID4B, EZH2, MACROD2, MLL2, each n=1) were prominent in these cases and virtually absent in non-recurrent cases (n=6) or other pediatric acute lymphoblastic leukemia cases (n=18). In relapse nucleotide variations were detected in cell fate determining transcription factors (GLIS1, AKNA). Structural genomic alterations affected genes regulating B-cell development (IKZF1, PBX1, RUNX1). Eleven novel translocations involved the genes ART4, C12orf60, MACROD2, TBL1XR1, LRRN4, KIAA1467, and ELMO1/MIR1200. Typically, patients harbored only single structural variations, except for one patient who displayed massive rearrangements in the context of a germline tumor suppressor TP53 mutation and a Li-Fraumeni syndrome-like family history. Another patient harbored a germline mutation in the DNA repair factor ATM. In summary, the relapse patients of our cohort were characterized by somatic mutations affecting the RAS pathway, epigenetic and developmental programs and germline mutations in DNA repair pathways