Co-Existance of JAKV617F and PIG-A mutations in Primary Budd-Chiari Syndrome.
Authors: Sugimori Chiharu , Shain Kenneth H , Caceres Gisela , Sokol Lubomir , Araten David , Maciejewski Jaroslaw P. , Zou JianXiang , Epling-Burnette P.K. , List Alan F 
Published: October 2019

Abstract
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 JAKV617F. The clinical syndromes of PNH and JAKV617F mutations often present as overlapping clinical syndromes raising the possibility that acquired JAKV617F 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 JAKV617F 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 JAKV617F activating mutation. Of the 21 cases examined, PNH with JAK2V617F 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 JAK2V617F 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, JAK2V617F 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 JAK2V617F 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 JAK2V617F /PIG-A mutations may contribute clinically to specific manifestations of splenic venous thrombosis or hepatic vein thrombosis in association with intravascular coagulation and myelodysplasia.

Disclosures
No relevant conflicts of interest to declare.