Abstract
The recently discovered JAK2V617F mutation provides a critical insight into the molecular pathogenesis of polycythemia vera and other myeloproliferative diseases (MPD). However, the mutations present in patients with wild type JAK2 have not been discovered, and the precise molecular consequences of JAK2 mutation have not been elucidated. We employed gene expression profiling to characterize the molecular phenotype of cells with the JAK2V617F mutation, to identify a distinct signature in cells without JAK2 mutations, and to refine a molecular taxonomy of MPDs. Using purified neutrophils from 70 patients with myeloproliferative diseases and 11 unaffected individuals, we performed gene expression profiling using oligonucleotide microarrays, sequencing of the JAK2 gene, quantitative genotyping by mass spectrometry and allele-specific quantitative PCR, and X-inactivation clonality assays. To reduce the confounding influence of normal neutrophils that are admixed with cells bearing disease-causing mutations, we examined the gene expression profiles of samples in which greater than 80% of JAK2 alleles bear the V617F mutation. PRV1, a previously identified marker of polycythemia vera, was powerfully overexpressed in neutrophils with a homozygous JAK2 mutation. In addition, cells with the JAK2 mutation had increased expression of a set of kinases, including JAK2, and decreased expression of a set of phosphatases. Cells that rely on JAK2 activation for clonal dominance may therefore derive a selective advantage from increased expression of the JAK2 gene. We next examined samples that have high clonality, and therefore relatively few normal neutrophils, but do not have a mutation in the JAK2 gene. These samples have a markedly different gene expression profile and overexpress a distinct set of kinases. The kinases overexpressed in cells with wild type JAK2 are candidates for further mutational analysis and are potential therapeutic targets. Utilizing these signatures and unsupervised analytical algorithms, the samples cluster according to their mutational status and the percentage of normal neutrophils in the sample. Our data demonstrate that the gene expression profiles of MPD samples are not uniform regardless of JAK2 genotype, implying that samples with and without JAK2 mutations have not activated the same pathway via alternative mechanisms. Moreover, we have identified a common signature in samples without JAK2 mutations that meets significance, indicating that one or a small number of mutations may play a critical role in these cells. Genotype and gene expression analyses are defining a molecular classification of myeloproliferative diseases with subtypes that have distinct therapeutic targets.
Molecular-genetic aspects of the diagnosis of myeloproliferative diseases demonstrated on the example of polycythemia vera.
