RUNX1-PDCD6 fusion resulting from a novel t(5;21)(p15;q22) chromosome translocation in myelodysplastic syndrome secondary to chronic lymphocytic leukemia

Abstract

Leukemic cells often carry chromosome aberrations which generate chimeric genes of pathogenetic, diagnostic, and prognostic importance. New rearrangements giving rise to novel fusion genes define hitherto unrecognized genetic leukemia subgroups. G-banding, fluorescence in situ hybridization (FISH), and molecular genetic analyses were done on bone marrow cells from a patient with chronic lymphocytic leukemia (CLL) and secondary myelodysplasia. The G-banding analysis revealed the karyotype 46,XX,del(21)(q22)[9]/46,XX[2]. FISH on metaphase spreads with a RUNX1 break apart probe demonstrated that part of RUNX1 (from 21q22) had moved to chromosome band 5p15. RNA sequencing showed in-frame fusion of RUNX1 with PDCD6 (from 5p15), something that was verified by RT-PCR together with Sanger sequencing. Further FISH analyses with PDCD6 and RUNX1 home-made break apart/double fusion probes showed a red signal (PDCD6) on chromosome 5, a green signal on chromosome 21 (RUNX1), and two yellow fusion signals, one on der(5) and the other on der(21). Reassessment of the G-banding preparations in light of the FISH and RNA-sequencing data thus yielded the karyotype 46,XX,t(5;21)(p15;q22)[9]/46,XX[2]. The t(5;21)(p15;q22)/RUNX1-PDCD6 was detected only by performing molecular studies of the leukemic cells, but should be sought after also in other leukemic/myelodysplastic cases with del(21q).

Fig 1. G-banding analysis of bone marrow from a patient with CLL and secondary MDS.

The initial analysis showed a del(21)(q22). Reassessment of the G-banding preparations after taking into account additional molecular-cytogenetic data showed a t(5;21)(p15;q22). Arrows indicate breakpoints.

Fig 2. FISH analysis of bone marrow from a patient with CLL and secondary MDS using a commercial RUNX1 break apart probe.

(A) Diagram illustrating the commercial RUNX1 probe. (B) Metaphase spread showing splitting of RUNX1. Most of the green signal has moved to distal 5p. (C) Ideograms showing the der(5)t(5;21)(p15;q22) and der(21)t(5;21)(p15;q22) together with their normal chromosome homologs.

Fig 3. Molecular genetic analysis of bone marrow from a patient with CLL and secondary MDS.

(A) Gel electrophoresis showing the amplified RUNX1-PDCD6 cDNA fragments. M, GeneRuler 1 Kb DNA ladder (Thermo Scientific). Lane 1, amplification using the primer set RUNX1-809N-F1/PDCD6-354R1. Lane 2, amplification with the primer set RUNX1-852N-F1/PDCD6-373R1. (B) Partial sequence chromatogram of the cDNA fragment showing the fusion (arrow) of RUNX1 and PDCD6. (C) The putative RUNX1-PDCD6 fusion protein. The Runt homology domain from RUNX1 is in green. The region with four EF-hand motives from PDCD6 is in yellow. The RUNX1-PDCD6 junction is in box.

Fig 4. FISH analysis of bone marrow from a patient with CLL and secondary MDS using home-made break apart/double fusion PDCD6 and RUNX1 probes.

(A) Ideogram of chromosome 5 showing the mapping position of the PDCD6 gene (vertical red line). (B) Diagram showing the FISH probe for PDCD6. Additional genes in this region are also shown. (C) Ideogram of chromosome 21 showing the mapping position of the RUNX1 gene (green box). (D) Diagram showing the FISH probe for RUNX1. Additional genes in this region are also shown. (E) FISH on metaphase plate with the PDCD6 (red signal) and RUNX1 (green signal) probes showing a red signal on chromosome 5, a green signal on chromosome 21, and two yellow fusion signals, one on der(5) and one on der(21). (F) FISH on interphase nuclei showing a red PDCD6 signal, a green RUNX1 signal, and two yellow fusion signals.