Polycythemia Vera with Multiple Clones Carrying Different Mutations (L611V, V617F, L611V/V617F) in Exon 14 of JAK2.

In more than 95% of cases, Polycythemia vera (PV) is characterized by the presence of the V617F mutation of JAK2 (JAK2-V617F). We report on three cases of V617F-positive PV with an additional mutation of JAK2 changing leucine 611 for a valine (L611V).

We used allele-specific quantitative PCRs and pyro-sequencing to detect the L611V and V617F JAK2 mutants in genomic DNA and in cloned PCR products. The consequences of the different mutations on the function of JAK2 were investigated using transient expression in BaF-3/EpoR cells.

We found equal proportions of L611V and V617F alleles in granulocyte DNA (patients Na249, 19%; Na382, 26%; Di362, 27%) and established the presence of alleles with L611V and V617F in cis (L611V/V617F). The double JAK2-L611V/V617F mutant represented 15% to 26% of JAK2 alleles in granulocyte DNA. JAK2-L611V/V617F was detected in 12% to 30% of erythroid colonies, always in a heterozygous fashion. Low levels of single mutant alleles were also found, in 0.5% of cloned PCR products (JAK2-L611V, patient Na249) and in 1.5% of genomic DNA and cloned PCR products (JAK2-V617F, patient Na249), indicating that mutations in JAK2 occurred more than once. Functionally, JAK2-L611V was found comparable to wild-type JAK2, whereas JAK2-L611V/V617F displayed greater constitutive tyrosine phosphorylation of JAK2 and AKT than wild-type JAK2 and JAK2-V617F. The double JAK2-L611V/V617F mutant was associated with a high hematocrit and normal counts of leukocytes and platelets. Patients Na382 and Di362, but not patient Na249, carried the 46/1 haplotype of chromosome 9p associated with a reported pre-disposition to mutations of JAK2 on the same allele.

PV patients can harbor multiple clones carrying different mutations of JAK2, which may or may not increase JAK2 activity. Functionally silent mutations, not leading to clonal expansion, may remain undetected. The double JAK2-L611V/V617F mutated clone was predominant whereas single L611V and V617F JAK2 mutants represented <2% of JAK2 alleles. Thus presence of the activating JAK2-V617F mutation does not ensure clonal expansion, implying that other factors promote or repress expansion of JAK2-mutated progenitors. Lastly, mutations of JAK2 can occur more than once in patients negative for the pre-disposing 46/1 haplotype.

No relevant conflicts of interest to declare.