Rearrangements of IER3 Represent a Novel and Recurrent Molecular Abnormality in Myelodysplastic Syndromes (MDS)

Background: For most patients with MDS, the disease-associated molecular abnormalities are unknown, which contributes to diagnostic uncertainty and has limited development of effective therapies. Chromosomal deletions such as del(5q) or monosomy 7 are more common than translocations in MDS, but balanced translocations are more likely to be pathobiologically informative. Here we report cloning of an MDS-associated translocation that revealed a novel recurrent molecular abnormality. Methods: We first studied a 71-year old man who presented with MDS and t(6;9)(p21.3;q34) as an isolated chromosomal abnormality in 18 of 20 metaphases. FISH assays for DEK/CAN and ABL rearrangements were unrevealing. We generated somatic hybrid murine/human cell lines from a buffy coat; microdissected and amplified derivative chromosomes; and hybridized to 6p/9q custom comparative genomic hybridization (CGH) arrays. The CGH results considerably narrowed the 6p and 9q breakpoints, which were then amplified by long-range PCR and sequenced to define the rearrangement. Results: The IER3 gene (Immediate Early Response 3; also known as IEX-1) at 6p21.3 was found to be separated from its upstream regulatory elements in this rearrangement and translocated to a transcript-poor region of 9q. RT-PCR confirmed marked downregulation of IER3 expression in the patient compared to healthy controls. IER3 is a plausible candidate for involvement in MDS because of its known role in regulating death receptor-induced apoptosis, interaction with NF-κb pathways, and its importance in response to genotoxic stresses such as ionizing irradiation. We then identified archival bone marrow cell pellets from 204 additional patients with various clonal hematological disorders and chromosomal rearrangements involving 6p21 or 6p22 (i.e., translocations, inversions, deletions, or additions), and designed a splitsignal FISH probe set to assay IER3. FISH results were abnormal in 8 additional patients (9/205 pts total abnormal, 4.4%): 3 split signals and 6 amplifications (one patient had both abnormalities); all 8 patients had MDS and 6p rearrangements as part of a more complex karyotype. FISH studies in MDS patients without 6p rearrangements are ongoing. RTPCR in MDS patients without 6p rearrangements demonstrated down-regulation of IER3 by >2-fold in 38% of patients (67% lower-risk) and up-regulation in 12% of patients (50% higher-risk) when compared to the mean for healthy controls. Direct sequencing of the IER3 coding region and the promoter region in 30 patients without 6p rearrangements revealed no point mutations, but the prevalence of non-synonymous single nucleotide polymorphism rs3094124 (p.A127P) was higher than expected in patients (variant in 28.3 % of MDS alleles vs. 8.6% of 116 HAP-CEU controls, p=0.0015). IER3−/− mice are known to have hypertension, cardiac hypertrophy, and epithelial proliferation, but are not anemic (Sommer SL et al J Appl Physiol 2006). We performed methylcellulose hematopoietic colony growth assays using bone marrow mononuclear cells from IER3−/− mice and wildtype controls, and noted no differences in BFU-E, CFU-E, and CFU-GM growth under unstressed conditions. Studies of IER3 expression induction under stressed conditions in primary MDS cells are ongoing. Conclusion: IER3 rearrangements represent a novel clonally-restricted recurrent genetic abnormality in MDS. Dysregulation of IER3 expression is common in MDS, even in patients without 6p rearrangements. This is the first time IER3 has been linked to human disease. Clarification of the role of IER3 in MDS is likely to yield new insights into MDS pathobiology.