Polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) can all be associated with the JAK2V617F gain of function mutation. MPDs are also associated with gross cytogenetic anomalies including genomic gain/loss or loss of heterozygosity (LOH) due to mitotic homologous recombination. To determine whether genomic anomalies may be associated with JAK mutation status/disease phenotype we subjected genomic DNA from the granulocytes of 87 patients with PV, ET or IMF to analysis using high resolution Affymetrix 250K Nsp SNP arrays with an average probe spacing of 12Kb. Firstly this analysis precisely mapped previously identified anomalies in MPD patients such as gain of chromosome 1q chromosome 9 (Chr9), chromosome 8, and deletion of 20q and 13q indicating the robustness of the technique. By removing genomic Copy Number Polymorphisms (CNPs) ascertained from 90 normal individuals from the HapMap dataset, we further detected a number of novel alterations such as frequent gain of Chr9p33–34 harboring among other genes, Notch1. MPD patients heterozygous for the JAK V617F mutation exhibited higher genomic instability than JAK2 V617 homozygous or wild type patients. IMF patients had overall more aberrations than PV or ET patients but unsupervised hierarchical clustering of chromosomal anomalies could not distinguish patients with PV, ET or IMF. Whole chromosomal gain or loss was equally frequent among the three diseases, however IMF patients had more frequent alteration of 10Mb or greater when compared to ET or PV (p<0.01) and ET and IMF patients both more frequently exhibited changes ranging from 1–10Mb in size than PV patients (p<0.01). Correlation of gene expression profiles from the granulocytes of 38 MPD patients with copy number profiles from DNA of the patients indicated that gene expression in regions of copy number gain or loss correlated with gene dosages in general. From 2493 genes residing in regions of copy number change region, 259 genes exhibited a positive correlation between copy number and gene expression. By calculating the t–statistics of expression values between samples with and without chromosomal aberrations we further identified 28 genes that showed a change of at least 1.5 fold (p<0.05), 27 of which were located on chromosome 9. Among these were Smarca2, NOTCH1 JAK2 itself. JAK2V617F expression was highly elevated by gain of Chr9 or Chr9p LOH. LOH on chr9p was detected in 16 patients, the majority of whom were confirmed as homozygous for JAK2V617F. To investigate how the over-expression of JAK2V617F might affect the expression of other genes and pathways, gene expression profiles of patients with Chr9 abnormality including Chr9 gain or Chr9p LOH were compared to samples with a normal copy number for Chr9. We identified 493 genes including 210 up-regulated and 283 down-regulated genes by at least 1.5 fold at a p value of 0.05 after multiple-testing correction. As a control gene expression patterns in JAK2-wildtype MPD granulocytes were compared to expression in normal granulocytes. This analysis identified an almost completely different set of genes; 441/493 (89%) of genes differentially expressed in Chr9 abnormal cases versus Chr9 normal cases were not found in the Chr9 normal versus normal control dataset. Up-regulated genes in Chr9 abnormal cases included CD177, CDK5RAP2, BIRC1, STAT5, TLR4 and down-regulated genes include KLRB1, Myc, RUNX3, P53CSV, Pathway analysis of dysregulated genes in Chr9 gain/LOH cases showed that genes involved in p38/MAP kinase, Toll-like receptor and Fc epsilon RI signaling were up-regulated and the genes involved in pathways of protein synthesis/translation and T cell receptor signaling were down-regulated. Collectively these findings suggest that MPD associated with Chr9 gain or 9pLOH and increased expression of JAK2V617F represents a distinct subset of MPD, characterized by less genomic instability than other cases. Furthermore, increased JAK2 and STAT5 expression and activity in these cases may induce distinct downstream pathways and alterations in gene expression.

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