JAK2 Genotyping (Real-Time Allelic Discrimination) Reveals Rare JAK2 V617F-Positive Erythroid Colonies in Pediatric Patients with Essential Thrombocythemia.

Childhood essential thrombocythemia (ET) is a very rare disease belonging to a group of Bcr-Abl negative myeloproliferative disorders. Our study involved 15 pediatric ET patients diagnosed at the age of 6–17 years. Monoclonal hematopoiesis was detected only in one patient, who is recently an adult (diagnosed at the age of 9). JAK2 V617F was detected in whole blood DNA by allele-specific (AS)-PCR and BsaXI restriction analysis. In our hands AS-PCR sensitivity reached the detection limit of one homozygous cell in 2000 normal cells. In addition, more sensitive AS-RT-PCR was performed on RNA isolated from granulocytes and platelets from peripheral blood. Only one patient was JAK2 V617F-positive as detected by these established protocols. In the clonal assay of hematopoietic progenitors, most patients showed hypersensitivity to erythropoietin (Epo) in vitro with the growth of Epo-independent erythroid colonies (EECs) in some of them. Over 300 myeloid colonies were picked and analyzed by real-time allelic discrimination assay for JAK2 V617F mutation run on a RotorGene 3000 instrument (Corbet Research, Australia) using fluorescently labeled LNA-modified probes. We have detected a few JAK2 V617F-positive EECs and CFU-GMs in 4 patients from our cohort. For example in one patient out of 7 analyzed EECs, one colony was homozygous and one heterozygous for the mutation. In the same patient out of 10 analyzed CFU-GMs one was homozygous for the mutation. In another patient out of 27 analyzed EECs only one was heterozygous. Some positive clones were confirmed by direct sequencing. Our data clearly show that some childhood ET patients posses JAK2 V617F-positive hematopoietic colonies derived from in vitro assays of committed hematopoietic progenitors. The rarity of the mutated subclones and the inability to detect them from peripheral blood using standardized protocols suggest either that the culture conditions without addition of Epo led to a great enrichment of the mutated colony growth or that the JAK2 V617F positive clones may be repressed in ET. The presence of JAK2 V617F clone in our cohort was not associated with any severe clinical symptoms. It would be interesting to follow up the patients for possible subsequent acquisition of JAK2 mutation as well as possible evolution of the disease to a clonal one or to polycythemia vera.

This work was supported by Ministry of Health of the Czech Republic (Grant NR9471-3), Ministry of Education, Youth and Sports of Czech Republic (projects MSN 6198959205 and MSN 6198959216).