Identification of a novel IGH‐MMSET fusion transcript in a human myeloma cell line with the t(4;14)(p16·3;q32) chromosomal translocation

We described a novel IGH-MMSET fusion transcript in the context of the molecular characterization of the recently established human myeloma cell lines (HMCLs) KMS26, KMS28BM and KMS34, found to express high levels of FGFR3 transcripts (Otsuki et al, 2002). We further confirmed this evidence by Western blot analysis showing expression of fgfr3 in all three cell lines, albeit at different levels. Activating mutations within specific domains of the FGFR3 gene were not found in all three cell lines by using the protocols previously described (Intini et al, 2001). Finally, KMS28BM expressed the fgfr3-IIIb isoform, similar to that reported in KMS18 (Ronchetti et al, 2001) (data not shown). The presence of t(4;14) was investigated by dual-colour fluorescence in situ hybridization using a combination of BAC clones specific for FGRF3, MMSET and IGH loci. Dissociation of FGFR3 and MMSET signals and association of FGFR3 and IGH loci were detected in all cell lines (data not shown). Analysis of IGH-MMSET fusion transcripts was performed by reverse transcription polymerase chain reaction (RT-PCR) as previously described (Malgeri et al, 2000). Fragments specific for the MB4-1 and MB4-3 type breakpoints were detected in KMS34 and KMS26, respectively, whereas a fragment smaller than the MB4-3 type breakpoint was found in KMS28BM. Its direct sequencing using the IGH and ms6r primers (respectively 54 and 108 bp in length) revealed the presence of MMSET exon 6 but the absence of the entire MMSET exon 5 (data not shown), thus strongly suggesting the occurrence of a novel breakpoint within intron 5 of the gene (Fig 1A). To characterize this novel transcript, we attempted the molecular cloning of the 4p16Æ3 breakpoint in KMS28BM as previously described (Richelda et al, 1997). A 10 kb BamHIrearranged JH fragment containing 4p16Æ3 sequences was isolated (Fig 1B) and the sequencing analysis of a HindIII fragment positive for an Sl probe revealed two particular findings. First, the Sl sequence was joined to an Sc1 sequence, thus suggesting that the recombination event occurred after a legitimate class switch recombination, a finding previously observed in an HMCL and two primary cases harbouring t(4;14) (Fenton et al, 2003). Secondly, the 4p16Æ3 sequence juxtaposed to Sc1 was derived from the 4p16Æ3 cosmid clone L75b9c (starting at nucleotide 554) (see scheme in Fig 1B). This breakpoint was therefore telomeric to the untranslated MMSET exon 1a, occurred 295 bp upstream of the KMS11 breakpoint (Richelda et al, 1997) (see schema in Fig 1C), and should have accounted for an MB4-1 type transcript. To clarify this finding, we investigated the arrangement of the genomic region containing MMSET exon 6 by Southern blotting. Rearranged fragments were detected with both EcoRI and BamHI restriction enzymes (Fig 1C). The cloning of the 12 kb EcoRI fragment demonstrated that MMSET intron 5 sequence ended 112 bp upstream of exon 6 (nucleotide 18729 of cosmid L190b4) (Fig 1C) and was juxtaposed to a sequence derived from the more telomeric cosmid L184d6 (starting at nucleotide 16069), thus indicating the occurrence of an internal deletion of 13141 nucleotides. Both breakpoint sites occurred within Alu repeat regions. We suggest that the 4p16Æ3 breakpoint in KMS28BM occurred within the 5¢ regulatory region of the MMSET gene, and was followed by an internal deletion involving translated exons 3–5, leading to the generation of a novel IGH-MMSET fusion transcript. However, putative truncated MMSET proteins generated by this transcript are similar to those predicted in t(4;14) cases harbouring the MB4-3 (Malgeri et al, 2000). Furthermore, this novel fusion transcript may represent a rare event as it was not detected in eight HMCLs and 26 primary MMs with t(4,14) that were investigated in our laboratory (Malgeri et al, 2000 and data not shown).