Figure 1
Figure 1. Molecular characterization of the two new MLLT10 fusions. (A) Two HNRNPH1-MLLT10 splicing isoforms were identified in patient 1. (Top) Direct sequencing showed an in-frame HNRNPH1-MLLT10 isoform joining nucleotide 1324 (HNRNPH1 exon 11) to nucleotide 2097 (MLLT10 exon 15). (Bottom) Cloning and sequencing showed nucleotide 6701 (HNRNPH1 intron 10) fused in-frame with nucleotide 2097 (MLLT10 exon 15). Hypothetical fusion protein was shown in which HNRNPH1 maintained all 3 RRM at the N terminus and MLLT10 lost 2 of 3 NLS. MLLT10 maintained the critical OM-LZ domain at the C terminus. Primer and sequence numbers refer to GenBank accession NC_000005.9, NM_005520.2, NP_005511.1 for HNRNPH1 and NM_004641.3, NP_004632.1 for MLLT10. (B) DCDF test: Probes for MLLT10 (RP11-418C1 and RP11-249M6) in orange and for HNRNPH1 (CTD-3223H16 and RP11-410B18) in green showed 1 fusion signal on der(10) (arrow). (C) Two DDX3X-MLLT10 splicing isoforms were identified in patient 2. (Top) Sequencing showed an in-frame DDX3X-MLLT10 isoform joining nucleotide 958 (DDX3X exon 2) to nucleotide 510 (MLLT10 exon 3). (Bottom) Cloning and sequencing showed an in-frame isoform with nucleotide 900 (DDX3X exon 1) fused with nucleotide 590 (MLLT10 exon 4). The hypothetical fusion protein lost the DDX3X DEAD box domain at the N terminus and maintained part of the PHD, all 3 NLS and the OM-LZ domain at the C terminus. Primer and sequence numbers refer to GenBank accession NM_001356.3, NP_001347.3 for DDX3X and NM_004641.3, NP_004632.1 for MLLT10. (D) DCDF-FISH with probes for DDX3X in green (RP11-1058N11, flanking 5′, and RP11-10K13, flanking 3′) and MLLT10 in red (RP11-418C1 and RP11-249M6), showed 1 fusion signal (arrow). (E) Unsupervised analysis of 11 T-ALL HOXA patients. In such unsupervised analysis, patients bearing MLLT10 rearrangements and those without MLLT10 rearrangements (1 MLL-ENL, 1 MLL-AF6, 2 TCRB-HOXA, and 1 SET-NUP214) are naturally clustered in 2 distinct groups. PICALM-MLLT10 patients are indicated in orange; patient 1 and patient 2 (HNRNPH1-MLLT10, DDX3X-MLLT10) in green and patients without MLLT10 rearrangements in blue. (F) Supervised analysis was created using the significative probe sets from the comparison of HOXA patients with MLLT10 rearrangements (4 with PICALM-MLLT10 and the 2 new cases with HNRNPH1-MLLT10, DDX3X-MLLT10) vs patients without MLLT10 rearrangements (1 MLL-ENL, 1 MLL-AF6, 2 TCRB-HOXA, and 1 SET-NUP214). Patients bearing MLLT10 recombinations are indicated in orange or green while patients without MLLT10 rearrangements are indicated in blue. DCDF, double-color double-fusion; LAP, Leukemia Associated Protein; NLS, nuclear localization signal; PHD, plant homeo domain; RRM, RNA recognition motif.

Molecular characterization of the two new MLLT10 fusions. (A) Two HNRNPH1-MLLT10 splicing isoforms were identified in patient 1. (Top) Direct sequencing showed an in-frame HNRNPH1-MLLT10 isoform joining nucleotide 1324 (HNRNPH1 exon 11) to nucleotide 2097 (MLLT10 exon 15). (Bottom) Cloning and sequencing showed nucleotide 6701 (HNRNPH1 intron 10) fused in-frame with nucleotide 2097 (MLLT10 exon 15). Hypothetical fusion protein was shown in which HNRNPH1 maintained all 3 RRM at the N terminus and MLLT10 lost 2 of 3 NLS. MLLT10 maintained the critical OM-LZ domain at the C terminus. Primer and sequence numbers refer to GenBank accession NC_000005.9, NM_005520.2, NP_005511.1 for HNRNPH1 and NM_004641.3, NP_004632.1 for MLLT10. (B) DCDF test: Probes for MLLT10 (RP11-418C1 and RP11-249M6) in orange and for HNRNPH1 (CTD-3223H16 and RP11-410B18) in green showed 1 fusion signal on der(10) (arrow). (C) Two DDX3X-MLLT10 splicing isoforms were identified in patient 2. (Top) Sequencing showed an in-frame DDX3X-MLLT10 isoform joining nucleotide 958 (DDX3X exon 2) to nucleotide 510 (MLLT10 exon 3). (Bottom) Cloning and sequencing showed an in-frame isoform with nucleotide 900 (DDX3X exon 1) fused with nucleotide 590 (MLLT10 exon 4). The hypothetical fusion protein lost the DDX3X DEAD box domain at the N terminus and maintained part of the PHD, all 3 NLS and the OM-LZ domain at the C terminus. Primer and sequence numbers refer to GenBank accession NM_001356.3, NP_001347.3 for DDX3X and NM_004641.3, NP_004632.1 for MLLT10. (D) DCDF-FISH with probes for DDX3X in green (RP11-1058N11, flanking 5′, and RP11-10K13, flanking 3′) and MLLT10 in red (RP11-418C1 and RP11-249M6), showed 1 fusion signal (arrow). (E) Unsupervised analysis of 11 T-ALL HOXA patients. In such unsupervised analysis, patients bearing MLLT10 rearrangements and those without MLLT10 rearrangements (1 MLL-ENL, 1 MLL-AF6, 2 TCRB-HOXA, and 1 SET-NUP214) are naturally clustered in 2 distinct groups. PICALM-MLLT10 patients are indicated in orange; patient 1 and patient 2 (HNRNPH1-MLLT10, DDX3X-MLLT10) in green and patients without MLLT10 rearrangements in blue. (F) Supervised analysis was created using the significative probe sets from the comparison of HOXA patients with MLLT10 rearrangements (4 with PICALM-MLLT10 and the 2 new cases with HNRNPH1-MLLT10, DDX3X-MLLT10) vs patients without MLLT10 rearrangements (1 MLL-ENL, 1 MLL-AF6, 2 TCRB-HOXA, and 1 SET-NUP214). Patients bearing MLLT10 recombinations are indicated in orange or green while patients without MLLT10 rearrangements are indicated in blue. DCDF, double-color double-fusion; LAP, Leukemia Associated Protein; NLS, nuclear localization signal; PHD, plant homeo domain; RRM, RNA recognition motif.

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