LAUSR

For nearly 3 decades, the human MLL (KMT2A) gene and its rearrangements have been investigated in many different laboratories around the world. At our diagnostic center (DCAL Frankfurt), our standard strategy for the identification of MLL-r is based on two independent approaches, namely “Multiplex” (MP)-polymerase chain reaction (PCR) and “Long distance inverse” (LDI)-PCR approach [1]. The MP-PCR approach is used to rapidly identify the eight most frequent MLL fusions (AF4, AF6, AF9, AF10, ENL, ELL, EPS15, and PTDs) which encompass ~90% of all diagnosed MLL-r leukemia patients, while LDI-PCR is used for all other patients (~10%). By applying both technologies, we have accumulated 94 direct MLL-gene fusions and 247 reciprocal fusion partner genes [2]. Nearly, all breakpoints have been identified in the major breakpoint cluster region (BCR) of the MLL gene (MLL exons 8–14). However, some of the patients remained negative, although they were positively prescreened by various methods. In order to diagnose MLL breakpoints in every patient, a total of 2688 overlapping Illumina capture probes covering the whole-MLL gene were designed and used to analyze a cohort of AL patients (n= 109) where we had either limited (n= 4; PCR positive but not sequenced) or no information (n= 105) on their molecular status. As depicted in Fig. 1a, we identified chromosomal rearrangements in 93 out of 109 patient cases. Sixteen patients remained MLL-r negative and were therefore assigned as patients with “unknown status”. The data analyses of the remaining 93 patients revealed the following distribution: for 67 patients (72%) a breakpoint could be analyzed in the major BCR; 5 patients (5%) displayed only the reciprocal der(TP) with breakpoints in exon 11 (putative CEP83-MLL spliced fusion), intron 11 (n= 3; putative FKBP8-MLL spliced fusion, AF9-MLL, RELAMLL) and intron 27 (IFT46-MLL), respectively. Surprisingly, an additional 21 patients (23%) had their breakpoints outside of the major BCR, but inside a novel, minor BCR. This novel BCR is localizing between MLL intron 19 and exon 24 (with a clear preference for MLL intron 21–23). Most of the new BCR cases represented MLL–USP2 gene fusions (n= 17). USP2 is localized about 1 Mbp telomer to MLL at 11q23.3 and transcriptionally orientated in direction of the centromere of chromosome 11, classifying all these fusions as intrachromosomal inversions (see Fig. 1b). In addition, we identified four balanced translocations in the minor BCR: one patient with an USP8 fusion (see Fig. 2 and Suppl. Figure S1), two with AF4 and one with AF9. MLL–USP2 and MLL–USP8 alleles seem to be restricted to the minor BCR (see Fig. 2), because they were never diagnosed in association with the major BCR. Most of the reciprocal USP2–MLL fusions were scattered over a larger region at 11q23.3 (see Fig. 2), involving also upstream (C2CD2L) and downstream genes (USP2-AS1). Our analysis revealed also five patients with 3′-MLL deletions that were caused by microdeletions (<200 bp), larger deletions (up to 34 kbp), or complex rearrangements including other These authors contributed equally: Claus Meyer, Bruno A. Lopes