A Molecular Cytogenetic Profile of the Chronic Myeloproliferative Disorders

The Chronic Myeloproliferative Disorders (CMPD) form a continuum of malignancies, the molecular pathogenesis of which remains obscure. Chronic Myeloid Leukaemia (CML) is the only disorder with a recognisable pathogenomic abnormality, the Ph chromosome. The remaining disorders have as yet, no defined disease markers although up to 30% display recurrent cytogenetic aberrations. Historically, conventional cytogenetic methods have relied on culturing of bone marrow (BM) aspirates to obtain suitable and sufficient mitotic figures for G-banded analysis. CMPD samples routinely have increased failure rates due to reduced growth and poor chromosome morphology. However, the application of Growth Factor (GF) stimulants to short term (24-48hr) BM cultures significantly improved both the quality and quantity of metaphases. BM samples were stimulated with three GF combinations namely. Bone Marrow Growth Supplement (BMGS), Myeloid Expansion Medium (MEM) and Conditioned Supernatant (CS) from human bladder carcinoma cell line 5637. To assess whether application ofGF stimulants leads to clonal selection, cultured samples from 15 patients were analysed by Fluorescence In situ Hybridisation (FISH), which supported the theory that clonal selection remained unaltered in stimulated samples. In addition to this, immunophenotyping of cells demonstrated that the lineage of propagated cells remained unaltered. By applying these improved techniques, CMPD samples and immortalised cell lines were then investigated for cryptic aberrations. Array comparative genomic hybridisation (CGH) investigations performed on CML cell lines revealed a complex pattern of imbalances affecting the short arm of chromosome 9, on the background of a 2-3 fold amplification. To clarify this observation a series of Bacterial Artificial Chromosome (BAG) Deoxyribonucleic Acid (DNA) probes sequenced to regions along 9p were selected. These probes were then applied to CMPD patient samples with both normal and abnormal karyotpyes, focusing on the region containing Janus Kinase 2 Gene (JAK2) at 9p24.1. In this ongoing study 18% of patients have been shown to carry a relative loss in this region. The pathogenomic significance of imbalances involving 9p remains unclear. It may however, represent an acquired homozygousity as a consequence ofmitotic recombination, with a “second hit” removing a wild type allele, leading on to further, as yet, un-clarified but clinically significant changes.

Name dc:type.qualificationname

PhD

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

De Montfort University

Year dc:date.issued

2007

Author dc:creator

• Vaughan, Beverley Rebecca

Licence

• https://dora.dmu.ac.uk/bitstreams/ae671317-dd5a-4ab5-8901-631c675eb370/download