Mutational analysis of class III receptor tyrosine kinases (C-KIT, C-FMS, FLT3) in idiopathic myelofibrosis Faisel M. Abu-Duhier, Anne C. Goodeve, Rory S. Care, Mamdooh Gari, Gill A. Wilson, Ian R. Peake and John T. Reilly Academic Unit of Haematology, Division of Genomic Medicine, Royal Hallamshire Hospital, Sheffield, UK Received 18 December 2001; accepted for publication 12 August 2002 Summary. Genomic DNA from patients with idiopathic myelofibrosis (IMF) was screened by polymerase chain reaction (PCR) and conformation sensitive gel electrophor- esis (CSGE) for mutations in the C-KIT gene (60 patients), as well as the C-FMS and FLT3 genes (40 patients). Intronic primers were used to amplify the entire coding region of both the C-KIT and C-FMS genes, and selected regions of the FLT3 gene. CSGE and direct DNA sequencing detected all previously reported as well as several novel polymorphisms in each of the genes. A novel c-fms exon 9 mutation (Gly413Ser) was detected in two patients. Its functional significance remains to be determined. The c-kit mutation Asp52Asn, previously described in two of six IMF patients in Japan, was not detected in this study. In addition, the reported c-fms mutations involving codons 301 and 969 were not identified. Therefore, in contrast to acute myeloid leukaemia, mutations in RTKs class III do not appear to play a significant pathogenetic role in idiopathic myelofibrosis. Keywords: c-kit, c-fms, FLT3, RTK class III, idiopathic myelofibrosis. Idiopathic myelofibrosis (IMF), or agnogenic myeloid metaplasia, is a chronic myeloproliferative disorder charac- terized by bone marrow fibrosis, extramedullary haemato- poiesis and a leuco–erythroblastic blood picture. The disease is a clonal haematopoietic stem cell disorder in which the fibroblast proliferation is a secondary, or reactive, phenom- enon, resulting from the inappropriate release of mega- karyocyte/platelet-derived growth factors, including platelet-derived growth factor (PDGF) and transforming growth factor-b (TGF-b) (reviewed by Reilly, 1999). Mega- karyocytes, however, may not be the only source for growth factors and it has been suggested that monocytic involve- ment may be pathogenetically important (Rameshwar et al, 1996). IMF is a preleukaemic disorder, with acute transforma- tions occurring in approximately 15% of patients (Siverstein et al, 1973). The malignant clone in IMF has a proliferative advantage, as revealed by an increased in vitro haemato- poietic progenitor cell growth, that results from either an enhanced sensitivity to growth factors or from an autolo- gous growth (Carlo-Stella et al, 1987; Han et al, 1988). The molecular events underlying the haematopoietic clonality are poorly understood. Retinoblastoma (RB) gene deletion (Morris et al, 1991) and impaired RB gene expression (Lebowitz et al, 1990) have been reported in individual patients, while RAS gene mutations only occur in 6% of patients (Reilly et al, 1994). More recently, Nakata et al (1995) reported c-kit mutations (Asp52Asn) in two of six patients and suggested that this acquired abnormality of the extracellular domain resulted in enhanced sensitivity of the patients’ stem cells to c-kit ligand (Kimura et al, 1997). c-kit is a member of the class III receptor tyrosine kinases (RTKs) that includes c-fms, FLT3, and the platelet-derived growth factor receptors a and b (Ullrich & Schlessinger, 1990). Class III RTKs share sequence homology and a similar overall structure with five immunoglobulin-like repeats in the extracellular domain, a single transmembrane domain (TM), a juxta-membrane domain (JM), two intracellular tyrosine kinase domains (TK1 and TK2) divided by a kinase insert domain (KI) and a C-terminal domain (Yarden & Ullrich, 1988). Mutations of this group of receptors have been linked recently to the pathogenesis of myeloid malignancies (Reilly, 2002). Internal tandem duplication mutations (ITD) of the FLT3 JM domain, for example, have been reported in approximately 20% of patients with acute myeloid leukaemia (AML) (Kiyoi et al, 1999; Abu-Duhier et al, 2000; Kottaridis et al, 2001) and appear to be the most important independent prognostic factor. FLT3 ITDs have also been reported in a small number of patients with myelodysplastic syndrome, but not in chronic myeloid Correspondence: Dr J. T. Reilly, Department of Haematology, H floor, Royal Hallamshire Hospital, Sheffield, S10 2JF, UK. E-mail: j.t.reilly@sheffield.ac.uk British Journal of Haematology, 2003, 120, 464–470 464 Ó 2003 Blackwell Publishing Ltd