Mutation and methylation analysis of the transforming growth factor beta receptor II gene in polycythaemia vera

Polycythaemia vera (PV) is a myeloproliferative disorder (MPD) thought to result from transformation of a haemopoietic stem cell. Transforming growth factor beta1 (TGF-beta1) is a negative regulator of haemopoietic stem cells, an effect mediated by direct binding to TGF-beta receptor II (TGF-beta RII). Reduced levels of TGF-beta RII mRNA or protein have been reported in several MPDs including PV, suggesting a role for TGF-beta RII in PV. No mutational analysis of the TGF-beta RII gene has yet been performed in PV. To investigate whether genetic or epigenetic alteration of the TGF-beta RII gene contributes to the pathogenesis of PV, we performed mutation and methylation analysis in 15 PV patients. The promoter, all seven exons and all intron/exon junctions were studied using single-strand conformation polymorphism (SSCP) and heteroduplex analysis (HA). In total, three single nucleotide polymorphisms (SNPs) were identified. These were located in the promoter, intron 2 and exon 5. No acquired mutations were detected in any patient sample. We also present a novel method, termed methylation-specific strand extension (MSSE), for the detection of methylated CpG dinucleotides. The combination of bisulphite modification and MSSE permits rapid analysis of the methylation status of CpG dinucleotides in multiple samples. We analysed the methylation status of the promoter and of a CpG island within exon 1 in 15 PV patients. No aberrant methylation was detected in either of these regions. These data demonstrate that neither mutation nor abnormal methylation of the TGF-beta RII gene is associated with the pathogenesis of PV. Furthermore, MSSE is a rapid and robust approach for assessing the methylation status of a given genomic region.