Definitive Chromosomal Orientation of the IL3-GMCSF to TCF-1 5q23 Region: Structural Analysis and Expression Profiles of a Novel Gene Encoding a Golgi-Associated Protein and of Two New Splice Isoforms Oo the Chondroitin Synthase 3 Gene Involved within the 5q Deletion in Ph(−) CML and MDS.
Authors: Kamakari Smaragda , Roussou Anastasia , Jefferson Andrew , Chatzidakis Ioannis , Ragoussis Ioannis , Anagnou Nicholas 
Published: October 2019

Interstitial deletion or loss of chromosome 5 [del (5q) or -5], is a frequent finding in myeloid leukemias and myelodysplasias (MDS) including a small subset of chronic myelogenous leukemia patients. Our group has focused on the 5q23–31 region encompassing the IL3-GMCSF and TCF-1 genes. To this end, we have initiated a systematic analysis of this subregion by generating a physical and transcript map employing a series of molecular and bioinformatics approaches. In our systematic search for genes involved in leukemogenesis, we have identified in the present study a novel gene and two novel splice variants of the chondroitin synthase 3 gene. The novel gene exhibits significant alternative splicing, generating at least seven splice variants encoding five putative proline-rich protein isoforms, sharing the N-terminal and central regions, while varying at the C-terminus. Interestingly, the novel protein contains several copies of the PXXP motif and one copy of the PPLP motif, a feature of proline-rich proteins interacting with SH3 and/or WW domains, respectively. The gene is ubiquitously expressed and conserved among species and may be involved in the Wnt signaling pathway. The subcellular distribution of one of the splice variants AJ427339, was investigated in transient expression experiments in HeLa cells. The GFP-AJ427339 fusion protein was localised to Golgi apparatus exhibiting additionally puncta in the cytoplasm, representing secretory organelles. Furthermore, we have isolated the complete cDNA sequences and of two novel isoforms of the chondroitin synthase 3 gene, representing splice variants and finely mapped it for the first time by FISH upstream of the IL3 gene. To further clarify the definitive orientation of this chromosomal region encompassing the IL3 and TCF-1 genes and delineate the order of the genes contained within, we performed two and three-colour FISH on metaphase chromosomes and interphase nuclei, using seven genomic clones. The data unambiguously established for the first time the definitive orientation of the region within the context of the long arm of chromosome 5, along with the order of the key regulatory genes, which is as follows: cen-AC005611[novel gene]-chondroitin synthase 3-IL3/GMCSF-RIL-AF5q31-TCF1-AC006077-tel. Finally, to further assess the significance of this region and its involvement in clonal myeloid disorders, we have performed a systematic FISH analysis to metaphase chromosomes, using eight genomic PAC and BAC clones as probes, covering a genomic region of 7 Mb in a case of a Ph(−) chronic myelogenous leukemia patient with a complex translocation between the two homologues of chromosome 5, as the sole aberration, leading to a deletion of 5q21 to 5q33 and on three MDS patients harboring 5q deletions defined by multicolor banding. The critical subregion containing the above genes, along with several putative transcriptional units of hematopoietic specificity, was found to be deleted in this atypical CML and in the three informative MDS patients, underscoring the significance of this subregion in the leukemogenesis process. These data provide the impetus for further functional studies to delineate the role of the individual regulatory genes contained within this subregion.