Second International Conference on Myelodysplastic Syndromes 7 (Cf9 MICRODELETION OF CSFIR IN MYELO- DYSPLASIA. J Boultwood, K Rack, J Madden, D OSCler, V Buckle and J S WalnSCOat. LRF Molecular & Cytogenetlc Haematology Unit, John Radcliffe Hospital, Oxford and Haematology Department, The Royal Victoria Hospital, Bournemouthi There is considerable interest in whether the deletion of one of the haemopoietic growth factor or receptor genes is critical to pathogeneis in the 5q- syndrome. We have carried out a molecular examination of the candidate gene CSFIR, both on the 5q- chromosome and on the apparently normal homologous chromosome 5, in 10 patients with myelodysplasia and a 5q deletion. Using restriction fragment length polymorphism analysis and gene dosage experiments all 70 ‘paiients showed deletion if CSF/Ry 6 oi 10 were hemizvaous and 4 of 10 homozvaous for CSNR loss. The homo;igous CSNR loss has bein confirmed in 2 patients by an in situ hybridization technique comparing the signal in affected cells to that in control sex-mismatched cells on the same slides. In those patients considered to have homozygous CSFlR loss by DNA experiments the gene was deleted from the 5q chromosome in all cells and from the apparently normal chromosome 5 in a subset of cells. This loss of one CSFlR allele, together with loss in some cells of the remaining allele on the homologous chromosome 5, in patients with myelodysplasia indicates that this is a region of critical gene loss on 5q. We have subsequently screened 58 patients with MDS and cytogenetically normal chromosomes 5, and found 7 of 58 to have a microdeletion encompassing CSFIR. The loss of CSFlR may be important in the pathogenesis of human myeloid leukaemia. (C9) DIRECT SEQUENCINo OF C-FM IN MYELO- DYSPLASTIC SYNDROME AND A&IL A PAGLIUCA, M THOMAS', K TOBAL, F FARZANEH', GJ MUFTI. Departrents of Haematology and Mol- ecular Medicine , King's College Hospital, London. The human FM proto-oncogene located at 5q33.3 encodes a 972 amino acid transmembrane glycoprotein which functions as the receptor for M-CSF. Although its' expression is normally associated with the monocyte-macrophage lineage it has been detected in human AML of diverse subtype. FMS genes found in feline sarcoma viruses possess constitutive kinase activity in the absence of ligand. In vitro studies have confirmed the importance of mutation at codon 301 and 969. To assess the role of such muta- tions in vivo we initially studied 41 cases with MDS or AML using PCR and hybridieation to allele specific oligonucleotides. We detected 0 cases with mutations (6 at codon 969). Using a novel direct PCR sequencing method we have been unable to detect these mutations in 4 of the 6 cases at codon 969. However we have detected a silent mutation in a percentage of cells at codon 966 (CCC to CCA, proline) in a case with AML M4 (with a 301 mutation by ASO). The inability to confirm our AS0 hybridisation findings is perplexing but may be related $0 the small clone size of the mutation. Further enrichment techniques may be needed to identify these lesions. Our finding of a silent mutation at codon 966 highlights one of the drawbacks of AS0 hybridisation in that unsuspected mutations close to the mutation of interest may abrogate the specificity of the technique. Further sequence data is required to assess confidently the importance of these mutations. (ClV STUDIES OF CLONALITY IN PATIENTS WITH REFRACTORY ANAEMIA AND CHROMOSOME DELETIONS G Abrahamson , J Boultwood, J Madden, S Kelly , D Oscler, K Rack, V Buckle and J S Walnscoat. Leukaemla Research Fund Molecular and Cytogenetlc Haematology Unit, John Radcliffe Hospital, Oxford, and Department of Haematology, The Royal Vlctorla Hospital, Bournemouth . We have used X-linked restriction fragment length polymorphism (RFLP) - methylation and gene deletion analyses to investigate the nature of the stem cell of origin of MDS in six women wilh refractory anaemia (RA) and either a partial deletion of the long arm of chromosome 5 or monosomy 7. All six patients showed gene loss in granulocyte but not T-lymphocyte fractions, indicating monoclonality of the granulocytes but not T-lymphocytes. To assess the possibility of the deletion being a late, myeloid-restricted event in the pathogenesis of MDS, we subsequently peflormed X- RFLP - methylation studies using the highly informative probe M27p, and also a probe specific for the phosphoglycerate kinase gene . These studies confirmed the monoclonality of the granulocytes and the polyclonality of the T-lymphocytes. In addition, X- RFLP- methylation studies on the B-lymphocyte fraction of one patient demonstrated polyclonality. The findings of this study indicate lack of Involvement of the lymphocytes of these patients , suggesting that the stem cell of origin may be restriied to the rnyeloid lineage in at least some cases of MDS. (C11) CLONALITY OF HEMATOPOIESIS IN PATIENTS WITH APLASTIC ANEMIA AND MYELO- DYSPLASTIC SYNDROMES H. van Kamp, J.E. Landegent, R. Willemze, R. Jansen, W.E. Fibbe. Lab. of Exp. Hematol., Dept. of Hematol., University Medical Center, Leiden, the Netherlands. To determine the clonal origin of hematopoietic cells in acquired aplastic anemia (AA) and myelodysplastic syndromes (MDS). we have analyzed patterns of X- chromosome inactivation using RFLP’s of the X-linked genes PGK, and HPRT and the X-linked probe M27/? in conjunction with their methylation patterns. Nineteen female patients (median age 29 years) with an initial diagnosis of AA and 21 female patients (median age 66 years) with MDS were screened. In total 14/40 (35%) patients were heterozygous for the PGK1-gene, 4/40 (10%) for the HPRT-gene and 31/37 (84%) for M27b; 35/38 (92%) showed a polymorphism for at least one of the probes. In 13/19 (68%) of the AA patients and in all MDS patients tested peripheral blood cells showed a monoclonal pattern after digestion with the methyla- tion-sensitive restriction enzyme HpaII. The 4 AA pa- tients studied at presentation all showed a monoclonal pattern. Four of 7 patients with AA from whom puri- fied cells were available, exhibited clonal hematopoiesis in both myeloid and lymphoid cells. In contrast, in the 3 MDS patients tested only myeloid cells were affected.