PMC

A. Fosmid selection and position of homology to chromosomes 15 and 17. B. Schematic representation of fosmid homology to the STROML1/PML locus and to the CDC6/RARA locus. C and D. Interphase FISH identifies a fusion event on der(17), consistent with ins(17;15). E and F. Interphase FISH identifies a fusion on der(15), consistent with ins(15;17). Probes were labeled with: Spectrum Orange, STROML1/PML; Spectrum Green, CDC6/RARA.

A and B. Cytomorphology of initial bone marrow biopsy stained with Wright-Giemsa. A. 10x amplification. B. 60x amplification. C. Metaphase cytogenetics of the dominant clone (46, XX, del(9)(q12q32), del(12)(q12q21), −6, −16, add(16)(p13.2), +2 mar [13 of 20 cells]). D. Metaphase cytogenetics of the minor clone (46, XX, del(9)(q12q32), del(12)(q12q21)[6 of 20 cells]). E. FISH using Abbott/Vysis dual fusion, dual probes. Note one fusion signal, two red signals (chromosome 15) and one green signal (chromosome 17). F. Interphase FISH using Abbott/Vysis dual fusion, dual probes. Note fusion on der(17). Probes were labeled with: Spectrum Orange, PML; Spectrum Green, RARA.

A. Schematic representation of ins(17;15) identified by WGS and resulting in PML-RARA fusion. Breakpoints are: chromosome 15: 72027045 and 72104113; chromosome 17: 35742679 and 35742683 (NCBI36/hg18). ATG: CDS start. ZF: zinc finger domain. CC: coiled-coil domain. DBD: DNA-binding domain. Arrows indicate binding sites of primers used in panel C. B. PCR validation of ins(17;15), del(12), del(14) and del(19). PCR of genomic DNA from the patient’s skin (normal: N) and leukemia (tumor: T) using primers that span the junction of RARA-LOXL1 (P1/P2), PML-RARA (P3/P4), del(12), del(14), LOXL1-PML (P5/P6), and del(19). Note amplification across fusion breakpoints in the leukemia sample (T), but not in the skin sample (N) for all but del(19) specific primer pairs. C. RT-PCR validation of PML-RARA expression. RNA prepared from cryopreserved leukemia cells was amplified with a forward primer in PML exon 3 and a reverse primer in RARA exon 3. DNA ladder: 1500, 800, 500, 300, 200, 150, 100, 50 base-pairs. D. Single nucleotide variants that occur in protein coding sequences and the percentage of each SNV allele within the total bone marrow population. Note two distinct clones, consistent with the original metaphase cytogenetics ().