The ASXL1 Gene Alterations in Patients with Myeloid Diseases and Chromosomal 20q Deletion

Deletion of the long arm of chromosome 20 - del(20q) - is a recurrent abnormality observed in various myeloid disorders, including myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), or acute myeloid leukemia (AML). It is a primary cytogenetic aberration, occurring often as a sole abnormality or the first abnormality in complex karyotypes, therefore it is assumed to play a key role in pathogenesis of myeloid malignancies. The proximal breakpoints of the deletion are consistently located in the 20q11.21 band, and the distal breakpoints span from 20q13.13 to band 20q13.33. In our previous study we showed a fusion of the ASXL1 and TSHZ2 genes resulting from an isochromosome of a deleted 20q in a patient with MDS (Brezinova et al Br J Haematol 2014). The ASXL1 gene is one of the most frequently mutated genes in myeloid disorders, mutations are generally associated with more aggressive course of the disease and poor clinical outcome. The aim of this study was to determine the frequency of ASXL1 breakpoints and/or ASXL1/TSHZ2 fusion in del(20q) cases.

Fluorescence in situ hybridizations (FISH) with locus specific probes for 20q11 and 20q12 regions (Abbott, Des Plaines, IL; Kreatech Diagnostics, Amsterdam, The Netherlands) confirmed the cytogenetically observed deletions of 20q in a cohort of 20 patients with myeloid malignancies (MDS 13x, MPN 3x, AML 2x, myelofibrosis 1x, thrombocytopenia 1x). In 15 patients deletion of 20q was a sole aberration, in 2 patients a variant of del(20q), an isochromosome of deleted 20q, was detected by FISH with a subtelomeric probe, Vysis ToTel 20p/20q (Abbott). Metaphase FISH mapping with a set of 7 bacterial artificial chromosome (BAC) probes (BlueGnome, Cambridge, UK) distributed in 20q11.21 and 20q13.2, with a chromosome-20-specific centromeric probe (Kreatech Diagnostics) as a control was used for determination of the breakpoints. Array comparative genomic hybridization (CytoChip Cancer 180K, BlueGnome) was performed on DNA samples of bone marrow cells of 11 patients with suspected ASXL1 gene deletion to find out the gene copy number variation.

A weak signal of RP11-358N2 BAC probe (30.93-31.12 Mb from telomere on the short arm, 20q11.21) was observed in 6 patients(30%), suggesting the proximal breakpoint of the deletion in the ASXL1 gene (30.95-31.03 Mb). However, the distal breakpoint in the TSHZ2 gene (51.80-52.11 Mb; 20q13.2) was found in one patient only, as previously reported. In 6 patients (30%) the signal of RP11-358N2 BAC probe was not present on the derivative chromosome confirming the ASXL1 gene deletion. In the remaining 8 patients the proximal breakpoint of the deletion was determined distally to the ASXL1 gene, in 3 patients into 9.3 Mb region between the MAPRE and the PTPRT genes. None but 1 patient had the distal breakpoint localized in the TSHZ2 gene. Three patients died in a group of 12 patients with ASXL1 gene alteration (deletion or partial deletion).

Using combination of molecular cytogenetic methods we proved that the extent of 20q deletions varied among the patients with a frequent proximal breakpoint in the ASXL1 gene. In our cohort, the ASXL1 gene was altered (deleted or partially deleted) in totally 60% of patients. The determination of the ASXL1 gene alteration in del(20q) cases may have a clinical and prognostic impact, but the relations with clinical data should be studied in a larger cohort of patients.

Supported by MHCR project for conceptual development of research organization 00023736, RVO-VFN64165/2012, and GACR P302/12/G157/1.