Novel MYCBP::EHD2 and RUNX1::ZNF780A Fusion Genes in T-cell Acute Lymphoblastic Leukemia

Abstract

Background/aim: T-cell acute lymphoblastic leukemia (T-ALL) is a rare malignancy characterized by proliferation of early T-cell precursors that replace normal hematopoietic cells. T-ALL cells carry non-random chromosome aberrations, fusion genes, and gene mutations, often of prognostic significance. We herein report the genetic findings in cells from a T-ALL patient.

Materials and methods: Bone marrow cells from a patient with T-ALL were examined using G-banding, array comparative genomic hybridization (aCGH), RNA sequencing, reverse transcription polymerase chain reaction (RT-PCR), Sanger sequencing, and fluorescence in situ hybridization.

Results: G-banding revealed del(1)(p34), add(5)(q14), trisomy 8, and monosomy 21 in the leukemic cells. aCGH detected the gross unbalances inferred from the karyotyping results, except that heterozygous loss of chromosome 21 did not include its distal part; 21q22.12-q22.3 was undeleted. In addition, aCGH detected a submicroscopic interstitial 7.56 Mbp deletion in the q arm of chromosome 19 from 19q13.2 to 19q13.33. RNA sequencing detected and RT-PCR/Sanger sequencing confirmed the presence of two novel chimeras, MYCBP::EHD2 and RUNX1::ZNF780A. They were generated from rearrangements involving subbands 1p34.3 (MYCBP), 19q13.2 (ZNF780A), 19q13.33 (EHD2), and 21q22.12 (RUNX1), i.e., at the breakpoints of chromosomal deletions.

Conclusion: The leukemic cells showed the heterozygous loss of many genes as well as the generation of MYCBP::EHD2 and RUNX1::ZNF780A chimeras. Because the partner genes in the chimeras were found at the breakpoints of the chromosomal deletions, we believe that both the heterozygous losses and the generation of the two chimeras occurred simultaneously, and that they were pathogenetically important.

Keywords: EHD2; MYCBP; MYCBP::EHD2; RNA-sequencing; RUNX1; RUNX1::ZNF780A; T-cell acute lymphoblastic leukemia; ZNF780A; cytogenetics; fusion gene.

Figure 1. G-banding analysis of the bone marrow cells of the T-ALL patient. A karyogram is shown, depicting the chromosome aberrations of the leukemic cells corresponding to the karyotype 46,XY,del(1)(p34),add(5)(q14),+8,-21. Arrows indicate breakpoints.

Figure 2. Array comparative genomic hybridization (aCGH) examination of the bone marrow cells of the T-ALL patient. (A) Genetic profile of whole genome showing trisomy for chromosome 8 and losses from parts of chromosomes 1, 5, 19, and 21. (B) The cytogenetic location, position on GRCh37/hg19 assembly, size (in Mbp), and gain/loss of the genetic imbalances are presented.

Figure 3. aCGH showing the deleted part of the p arm of chromosome 1. (A) Based on the hg19 assembly the deletion ended at position chr1:39311945, on the subband 1p34.3. The most distal (p-telomeric) probe in the assay mapped at position chr1:10478. (B) The area at position chr1:39311945 hosting the genes Ras related GTP binding C (RRAGC), MYC binding protein (MYCBP), gap junction protein alpha 9 (GJA9) and rhomboid like 2 (RHBDL2). Because there were no probes on MYCBP and GJA9, the breakpoint could not map more precisely. Highlights indicate the deleted (loss) part.

Figure 4. aCGH showing the deleted part of the q arm of chromosome 5. (A) The deletion started at position chr5:90488653 on the subband 5q14.3 and ended on subband 5q35.3. The most distal (q-telomeric) probe in the assay mapped at position chr5: 180787863. (B) The area at position chr5:90488653 shows that the deletion is just downstream of the adhesion G protein-coupled receptor V1 gene (ADGRV1, also known as GRP98). Highlights indicate the deleted (loss) part.

Figure 5. aCGH analysis showing the interstitial deletion in q arm of chromosome 19. (A) Genetic profile of whole chromosome 19 showing the deletion started at position Chr19:40662574 on subband 19q13.2 and ended at chr19: 48223904 on subband 19q13.33. (B) The area at position Chr19:40662574 showing that the deletion started between the genes zinc finger protein 780A (ZNF780A) and mitogen-activated protein kinase 10 (MAP3K10). (C) The area at position chr19: 48223904 showing that deletion ended within the EH domain containing 2 (EHD2) gene. Because of the low number of probes at the breakpoint regions, the interstitial deletion in 19q13 could not be mapped more precisely. Highlights indicate the deleted (loss) part.

Figure 6. aCGH analysis showing the deleted part of chromosome 21. (A) The deletion ended at position Chr21: 36299935 on the subband 21q22.13. The most distal probe on the p arm of chromosome 21 in the assay mapped at position Chr21:10773805. (B) The area at position Chr21: 36299935 showing that the deletion ended within intron 2 of RUNX1.

Figure 7. Sanger sequencing and fluorescence in situ hybridization (FISH) of the bone marrow cells of the T-ALL patient. (A) Partial Sanger sequencing chromatogram showing the junction between exon 4 of MYCBP and exon 2 of EHD2. (B) Partial Sanger sequencing chromatogram showing the junction between exon 2 of RUNX1 and exon 3 of ZNF780A. (C) FISH analysis on metaphase plates using in-house prepared probes for the MYCBP (red labeled) and EHD2 (green label) genes showed a red signal corresponding to normal MYCBP on chromosome 1, a green signal on normal chromosome 19 corresponding to EHD2, a fusion red/green signal on der(1) chromosome which corresponded to the MYCBP::EHD2 chimera, and a red signal on der(19) indicating that material from chromosome bad 1p34 translocated to q13 of der(19).

Figure 8. The coding part of the chimeric MYCBP::EHD2 transcript. MYCBP sequence is shown in grey background. The domain which binds to N-terminal part of MYC and stimulates MYC activation is written with red letters. The nuclear exit signal is shown in light blue background. The EF hand motif and EPS homology domain are written with dark blue and purple letters and are shown in dark yellow background.