Figure 1.
Figure 1. The detection of a novel MLL rearrangement and combined analysis of morphology and FISH of the bone marrow cells in the myeloid lineage. (A) Partial karyotype (G-banding) for the t(11;11)(q13;q23). Both chromosomes 11 are shown. The arrows indicate the breakpoints in the rearranged chromosomes. (B) Genomic DNA of the patient analyzed by the long-distance inverse–polymerase chain reaction (LDI-PCR) method. BamHI-digested and religated genomic DNA was tested with 5 oligonucleotides in 4 different combinations6 (lane 1: A-B, lane 2: A-C, lane 3: A-D, and lane 4: A-E; M indicates marker). The analysis included a positive control using the oligonucleotides B and F (lane 5) that amplifies a 7.9 kb DNA of the MLL breakpoint cluster region. In the analysis of the patient, non-germline amplimers can be observed (white arrows). Such non-germline amplimers were isolated. Sequence analysis of the isolated bands identified the novel MLL partner gene ARHGEF17. (C) Reverse transcriptase (RT)–PCR analysis of MLL-ARHGEF17 fusion transcript. RT-PCR analysis of the immediate breakpoint region in bone marrow cell DNA of the patient amplifying a product of 222 bp with the following primers: forward primer sequence 5′-GTCTGTTGTGAGCCCTTCC-3′, reverse primer sequence 5′-CTGCATGTAGCCCTGC ATC-3′. Lane 1: MLL-ARHGEF17, primer concentration 3.3 nmol/mL; lane 2: MLL-ARHGEF17, primer concentration 10 nmol/mL; lane 3: positive control (EWS FLI1); lane 4: negative control, M indicates marker. (D) Combined analysis of morphology and FISH. The left panels (i, iv, vii) show the cells stained with May-Grünwald-Giemsa. The middle panels (ii,v,viii) display the same cells after FISH; image acquisition was performed using a green/orange dual filter set. The right panels (iii,vi,ix) show acquisition of the same cells using a single green filter. The hybridization pattern for the nuclei without the MLL rearrangement is 2 yellow signals with the green/orange dual filter set and 2 spaced green signals with a single green filter. The hybridization pattern for the nuclei with the MLL-ARHGEF17 rearrangement is one yellow/one red signal, where the yellow signal represents the normal intact MLL gene and the green signal the rearranged MLL gene. Two closely located green signals are detected using a single green filter. The top row shows a myelocyte (i) with 2 nonrearranged MLL signals (ii,iii), the middle row shows a promyelocyte (iv) with the MLL-ARHGEF17 rearrangement (v,vi), and the bottom row shows a “band” (vii) with the MLL-ARHGEF17 rearrangement (viii,ix). Image acquisition was performed as described previously9 with the Duet system (BioView, Rehovot, Israel) at × 1000 magnification.

The detection of a novel MLL rearrangement and combined analysis of morphology and FISH of the bone marrow cells in the myeloid lineage. (A) Partial karyotype (G-banding) for the t(11;11)(q13;q23). Both chromosomes 11 are shown. The arrows indicate the breakpoints in the rearranged chromosomes. (B) Genomic DNA of the patient analyzed by the long-distance inverse–polymerase chain reaction (LDI-PCR) method. BamHI-digested and religated genomic DNA was tested with 5 oligonucleotides in 4 different combinations6  (lane 1: A-B, lane 2: A-C, lane 3: A-D, and lane 4: A-E; M indicates marker). The analysis included a positive control using the oligonucleotides B and F (lane 5) that amplifies a 7.9 kb DNA of the MLL breakpoint cluster region. In the analysis of the patient, non-germline amplimers can be observed (white arrows). Such non-germline amplimers were isolated. Sequence analysis of the isolated bands identified the novel MLL partner gene ARHGEF17. (C) Reverse transcriptase (RT)–PCR analysis of MLL-ARHGEF17 fusion transcript. RT-PCR analysis of the immediate breakpoint region in bone marrow cell DNA of the patient amplifying a product of 222 bp with the following primers: forward primer sequence 5′-GTCTGTTGTGAGCCCTTCC-3′, reverse primer sequence 5′-CTGCATGTAGCCCTGC ATC-3′. Lane 1: MLL-ARHGEF17, primer concentration 3.3 nmol/mL; lane 2: MLL-ARHGEF17, primer concentration 10 nmol/mL; lane 3: positive control (EWS FLI1); lane 4: negative control, M indicates marker. (D) Combined analysis of morphology and FISH. The left panels (i, iv, vii) show the cells stained with May-Grünwald-Giemsa. The middle panels (ii,v,viii) display the same cells after FISH; image acquisition was performed using a green/orange dual filter set. The right panels (iii,vi,ix) show acquisition of the same cells using a single green filter. The hybridization pattern for the nuclei without the MLL rearrangement is 2 yellow signals with the green/orange dual filter set and 2 spaced green signals with a single green filter. The hybridization pattern for the nuclei with the MLL-ARHGEF17 rearrangement is one yellow/one red signal, where the yellow signal represents the normal intact MLL gene and the green signal the rearranged MLL gene. Two closely located green signals are detected using a single green filter. The top row shows a myelocyte (i) with 2 nonrearranged MLL signals (ii,iii), the middle row shows a promyelocyte (iv) with the MLL-ARHGEF17 rearrangement (v,vi), and the bottom row shows a “band” (vii) with the MLL-ARHGEF17 rearrangement (viii,ix). Image acquisition was performed as described previously9  with the Duet system (BioView, Rehovot, Israel) at × 1000 magnification.

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