| Primary myelofibrosis (PMF), as with other myeloproliferative disorders, results from an altered blood stem cell which has acquired a clonal proliferative and survival advantage over normal cells. This altered progenitor cell is known to be just below the cross-junction of the lympho-myeloid lineage commitment, at the level of myeloid cell differentiation, and primarily results in an abnormal proliferation of: megakaryocytes, platelets, erythrocytes (RBCs), granulocytes, and monocytes/macrophages.;Considerable effort in cancer research has been directed towards the identification of cancer molecular biomarkers which can better shape and stratify prognostic value of clinical and patho-biological features of risk of disease progression. Small single-nucleotide variations (SNVs) in the genome can provide mechanistic evidence for disease. However, identifying specifically relevant alterations for a disease is confounded by the enormity of non-specific or passenger SNVs which are represented in the highly heterogeneous human genome. Detection of larger gene-fusion associated genetic alterations, involving chromosomal translocations or rearrangements in a disease have a higher probability of prognostic value.;Until now, identification of cytogenetically detectable aberrations have been useful in understanding the initiation and progression of cancer and in directing molecular genetic approaches, including development of molecular targeted therapy. The molecular pathogenesis of BCR/ABL1-negative myeloproliferative neoplasms, including primary myelofibrosis remains poorly understood despite the 2005 discovery of JAK2V617F, a gain-of-function mutation present in a large majority of patients.;Recent advances in Next Generation Sequencing provide powerful tools for probing the human genome and present significant opportunities for researchers to gain a better understanding of mechanisms of disease. In particular, relatable diseases to those known to contain an exclusive DNA alteration resulting in disease-specific biology, like primary myelofibrosis, are excellent candidate diseases to interrogate with this technology.;Our objective for this work was to utilize a powerful high resolution mate-pair sequencing (HRMPS) technology along with a unique algorithm to identify DNA alterations in primary myelofibrosis. We initially hypothesized that by using a higher resolution and innovative platform, we could discover recurrent translocations or micro-DNA alterations that may play a role in the pathogenesis of primary myelofibrosis, a disease which has very limited treatment options.;Our results show the power of a high resolution mate-pair sequencing platform in identifying the molecular heterogeneity in patients with primary myelofibrosis and its ability to uncover submicroscopic deletions/translocations/fusions, and the precise mapping of breakpoints in those with overt cytogenetic abnormalities. This technique also confirms the genetic heterogeneity of PMF, given the low frequency of recurrent specific abnormalities, identified by this screening strategy.;We have additionally shown genetic complexity in one case of polycythemia vera (PV) evolving to post-polycythemic myelofibrosis (PPMM), and identified a novel gene fusion SETBP1/GTF2H3 which structurally resembles known fusion proteins and could potentially be involved in the pathogenesis and transformation of the disease.;We next characterized the expression of a gene we found in an overt, recurrent cytogenetic alteration, del(20)(q11.2-13.2) frequently seen in myeloproliferative neoplasms (MPNs), in order to consider the contribution of this gene to the disease. At the fusion of this breakpoint was the known tumor suppressor plant homeodomain finger 20 (PHF20), which is partially deleted as a result of the alteration. We discovered that not only is PHF20 expression significantly decreased in PMF relative to normal, PHF20 expression is also significantly decreased relative to PV, which has been shown to evolve into PMF. Finally, we reveal that the levels of PHF20 expression correlate with JAK2V617F expression in PMF. Our findings indicate a potential contribution of the tumor suppressor PHF20 to primary myelofibrosis, and PHF20 expression level may be involved in the progression from PV to PMF.;Finally, we have discovered a novel gene fusion with an overall frequency of 18% in MPNs, INTS3/CHTOP. We also demonstrate that this fusion provides a proliferative advantage when over-expressed in murine hematopoietic stem cell re-plating experiments in vitro which suggests it may have a potential functional pathogenic role.;Our discoveries have revealed the effectiveness and immediate importance of this technology in characterizing the genomic landscape of PMF. It will soon be possible to characterize all PMF patients utilizing this technology, as the incorporation of next generation sequencing, including HRMPS, is quickly becoming more available, cost effective, and easy to solicit. By incorporating this technology, we can increase the resolution in screening for potential key molecular alterations, and in the future this may suggest the best therapeutic interventions based upon the specific alterations observed. By combining this with new and novel therapies suggested by the specific alterations observed by mate-pair sequencing, we may have a new paradigm for monitoring and clinical treatment of this disease. |
