PF653 TARGETED NEXT‐GENERATION SEQUENCING IDENTIFIES NOVEL GENE VARIANTS IN JAK2V617F NEGATIVE PATIENTS WITH ERYTHROCYTOSIS AND JAK2 GGCC_46/1 HAPLOTYPE

Background: The aetiology of erythrocytosis is complex; it can be classified as primary or secondary and acquired or congenital. Primary erythrocytosis is a bone marrow disorder caused by an intrinsic genetic defect in the erythroid progenitor cell. Secondary erythrocytosis is caused by factors independent of the erythroid compartment, usually causing an increased erythropoietin production. To date, several studies have been performed on large patient cohorts to identify any clinical or laboratory feature helping to distinguish familial and congenital erythrocytosis or primary acquired forms such as polycythemia vera. However, the differential diagnosis of erythrocytosis patients remains complex. Aims: The aim of this study was to analyze a group of 15 patients with erythrocytosis by a targeted next generation sequencing (NGS) approach with a customized panel of 26 myeloid genes. All cases were negative for the classic JAK2 V617F mutation, but were characterized by the JAK2  GGCC_46/1 haplotype, a germline combination of polymorphisms known to be associated with the onset of myeloproliferative neoplasms. Methods: A targeted NGS approach by Ion Torrent sequencing was employed using a customized panel including 26 genes involved in myeloid malignancies. This gene panel was created using the Ion AmpliSeq Designer (Thermo Fisher). Selected variants were investigated for a potential pathogenetic role using the SIFT and Polyphen scores and the Catalogue of Somatic Mutations in Cancer database. Results: Overall, 25 genetic variants in 13 myeloid genes were identified in all 15 patients within the examined cohort. According to their frequency in the healthy population and to a germline or somatic nature in our patient series, these variants were divided into three different groups: polymorphisms, rare germline variants, and rare somatic variants. Overall, seven polymorphisms attributed a benign clinical significance, and having a frequency of 10–40% in healthy individuals, were detected at higher frequency in our patient cohort in the following genes: JAK2 (rs2230724, rs2274649, rs2230722) ANKRD26 (rs7897309), GATA2 (rs2335052), TET2 (rs34402524), and CALR (rs1049481). In addition to polymorphisms, our analysis revealed rare germline and somatic variants in the TET2, KIT, MPL, DNMT3A, FLT3, EZH2, JAK2, ASXL1, ANKRD26 , ZRSR2, and RUNX1 genes. At least one rare germline or somatic variant was detected in 13 of the 15 cases (87%), the most frequently mutated genes being TET2  (7/15, 47%), KIT (4/15, 27%), MPL and DNMT3A (3/15, 20%). Non canonical JAK2 K1055R and MPL V114 M variants, mapping in JAK2 exon 23 and MPL exon3, respectively, were identified in 3 patients, one patient showing both. Summary/Conclusion: In this NGS study, patients with erythrocytosis lacking known pathogenic driver mutations were almost all (87%) found to be positive for rare variants in myeloid genes. The large number of rare germline variants together with polymorphisms in the JAK2, ANKRD26, GATA2, TET2 , and CALR genes is in accordance with the co‐occurrence of the JAK2 GGCC germline haplotype, that seems to predispose to an increased risk of developing erythrocytosis. However, the pathogenic contribution of each identified new gene variant warrants further investigations.