Identification of PVT1-NSMCE2 in the leukemic patient cells. (a) G-banding analysis. Arrow indicates two marker chromosomes (mars). (b) SKY analysis for the patient identified two mars derived from chromosome 8 (arrows). (c) Copy number changes at 8q24 detected by high-resolution oligonucleotide array. NSMCE2, TRIB1, MYC, PVT1, CCDC26, GSDMC, and FAM49B are amplified. The direction of the arrows reflects the direction of gene transcription. NG: no gene. (d) Detection of three PVT1-NSMCE2 fusion transcripts by RT-PCR. Primers were P1S and NSMCE2-Ex4AS for 5'-PVT1-NSMCE2-3'. Lane Pt.1: leukemic cells from the patient; lane N: water; lane M: size marker. (e) Sequence analysis of NSMCE2 fusion transcript in the patient. (f) FISH finding of the patient using PVT1 probe. Multiple red signals indicate extrachromosomal amplification of 5’PVT1 on dmins. Co-localized red and green signals indicate normal PVT1. Inset shows 5’PVT1 amplification in a micronucleus equivalent of mar (arrow). (Additional file 5: Figure S3) (g) FISH finding from the patient using an NSMCE2 probe. Intense yellow signals indicate amplification of NSMCE2 on mars and co-localized red and green signals signify normal NSMCE2 on chromosome 8. Inset shows NSMCE2 amplification in a micronucleus equivalent of mar (arrow). (h and i) DAPI pictures of metaphase cells corresponding to (f) and (g). Arrows indicate mars. In metaphase, NSMCE2 amplification was detectable on mars. 5’PVT1 amplification were observed on dmins, however, PVT1 FISH probe sets could not identify mars because of the background dmins (f and h). (j) Results of LDI-PCR. Primers were NSM38374 and NSM38666 for 5'-PVT1-NSMCE2-3'. Lane Pt.1: leukemic cells from the patient; lane N: water; lane M: size marker. (Additional file 4: Table S2) (k) Genomic mapping of PVT1 and NSMCE2 exons and breakpoint. White vertical boxes represent exons; dotted line represents breakpoint of PVT1 and NSMCE2 in the patient detected by LDI-PCR. Horizontal line indicates the location of miRNAs.