Co-Existance of JAK^V617F and PIG-A mutations in Primary Budd-Chiari Syndrome.

Abstract 3193

Poster Board III-130

Paroxysmal nocturnal hemogloginuria (PNH) is a clonal genetic disorder associated with multiple mutations in the X-linked phosphatidylinositol glycan class A (PIG-A) gene that causes glycosyl phosphatidylinositol (GPI) anchor protein (AP) deficiency in patients with PNH. Conditional PIG-A gene inactivation (PIG-A (-/-)) in hematopoietic cells of mice does not induce overt proliferative advantage to the HPC compartment. In humans, the molecular basis of clonal expansion of GPI-AP deficient cells is unknown but may relate to selective pressure from the bone marrow microenvironment conducive to GPI-AP survival. Alternatively, coexistence of additional genetic anomalies that confer growth potential has been speculated. A single mutation in the Janus Kinase (JAK)-2 gene has been reported to be the underlying molecular mechanism for myeloproliferative diseases including polycythemia vera (PV), essential thrombocythemia, and myelofibrosis. Substitution of a valine for phenylalanine destabilizes the auto-inhibitory activation domain of JAK2 leading to myeloproliferation. Genetic evidence and in vitro functional studies indicate that V617F gives hematopoietic precursors proliferative and survival advantages. A high proportion of patients with myeloproliferative disorders and myelodysplastic syndrome (MDS) carry this dominant gain-of-function mutation of JAK^V617F. The clinical syndromes of PNH and JAK^V617F mutations often present as overlapping clinical syndromes raising the possibility that acquired JAK^V617F mutations confer growth and survival potential to PNH clones in at least a subset of patients. To address this possibility, 21 PNH patients were screened for the presence of JAK^V617F mutations. We show here that the features of Budd-Chiari syndrome (BCS) as well as a predisposition to myelodysplastic syndrome can be caused by an acquired co-existing PIGA and JAK^V617F activating mutation. Of the 21 cases examined, PNH with JAK2^V617F mutations co-existed in three cases (14.3%). Thus far, two patients have been well characterized. Both cases were male, ages 51 (case 1) and 65 year-old (case 2) with normal cytogenetics and de novo classic PNH manifested by Budd-Chiari syndrome. Flow cytometry assay using CD55, CD59, and CD48 (GPI-APs) and FLAER to detect PNH type cells showed that 90% of granulocytes (case 1) and almost 100% of granulocytes (case 2) were GPI-AP deficient cells. Interestingly, T-cells in both cases displayed a normal phenotype suggesting that the PIGA gene mutation was selectively acquired in a common myeloid progenitor (CMP). From sorted populations of PNH+ and normal type granulocytes (case 1 only) or T cells (case 2), the JAK2^V617F mutant clone was found to co-exist with the PIGA mutation using allele-specific polymerase chain reaction (PCR). Real-time quantitative PCR using primers and probe to the junction of exon 5-6 coding region demonstrated drastically low to absent mRNA transcript expression of PIGA only in PNH-type granulocytes in both cases. Individual RT-PCR of the coding region of each exon (2-6) confirmed that transcription was absent in case 1 but limited to regions encoded by exons 2,4, and 6 in case 2. These results suggest that case 2 possesses a splicing mutation involving exon 5 to 6. In conclusion, JAK2^V617F mutation is known to cause the constitutive activation of the JAK-STAT signaling pathway, and leads to autonomous cell growth in a cytokine-independent expansion of HPCs. While it has been reported that JAK2^V617F mutation occurs in roughly 50% of primary Budd-Chiari syndrome and in 15 patients of 163 (9.2%) (Hoekstra et al, Journal of Hepatology, 6:19, 2009) of patients with PNH, these findings implicate co-existing JAK2^V617F /PIG-A mutations may contribute clinically to specific manifestations of splenic venous thrombosis or hepatic vein thrombosis in association with intravascular coagulation and myelodysplasia.