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Report on two novel nucleotide exchanges in the JAK2 pseudokinase domain: D620E and E627E

The V617F point mutation of the JAK2 gene (located in chromosome 9p24) shows a high frequency in diverse BCR-ABL-negative chronic myeloproliferative disorders. A thymidine>guanine substitution at position 1849 leads to a valine to phenylalanine exchange at amino acid 617, which is localized in exon 12. The highest frequency of point mutations of the JAK2 gene is found in polycythemia vera (80–95% of all cases), which is followed by essential thrombocytosis and idiopathic osteomyelofibrosis (40–55% of all cases). The mutation is further known to occur at low frequency in the so-called unclassifiable MPS (according to the WHO classification), in chronic myelomonocytic leukemia, in myelodysplastic syndrome and in acute myeloid leukemia (AML).1, 2, 3, 4, 5 The cytoplasmic nonreceptor Janus kinases regulate tyrosine phosphorylation of several essential signaling pathways for cell proliferation and apoptosis, for example, the JAK/STAT pathway. The V617F mutation induces hypersensitivity to cytokines or even independence from their influence in mutated cell lines and is conceived as gain-of-function mutation.1, 3, 6 It is situated in a highly conserved region of the JH2 pseudokinase domain of the JAK2 nonreceptor tyrosine kinase. The pseudokinase domain is involved in autoinhibition of the JAK2 tyrosine kinase activity by interaction of the amino acid 618 and surrounding residues with the activation loop of the JH1 kinase domain.1, 6, 7

All known reported point mutations of the JAK2 kinase in hematologic disorders represent the identical t1849g point mutation. The only exception is represented by a novel K607N mutation in a case of AML that had recently been reported by Lee et al.5 The NCBI single nucleotide polymorphism (SNP) database contains 435 SNPs of the JAK2 kinase ( We here report on two novel single nucleotide exchanges located in the JAK2 pseudokinase domain.

Polymerase chain reaction (PCR) was carried out in a 20 μl reaction volume with each 0.5 μ M of forward and reverse primer, 0.75 μ M Hyb-Probes, 4 mM MgCl2 and 2 μl LightCycler-FastStart DNA Master Hybridization Probes (Roche Diagnostics, Mannheim, Germany). LightCycler data were analyzed using the LightCycler 3.0 software (Roche Diagnostics, Mannheim, Germany). Each 20 μl reaction contained 2 μl of cDNA. Amplification was performed with 40 cycles using annealing temperature of an 60°C. Final melting-curve analysis was started at 40°C up to 85°C with slope of 0.2°C/s and continuous detection with channel F2/F1. Analysis for JAK2 V617F was performed with a melting curve-based LightCycler assay (forward primer JAK2-F: IndexTermAAGCAGCAAGTATGATGAG, reverse primer JAK2R: IndexTermCCCATGCCAACTGTTTAG, hybridization probes JAK2-A: IndexTermAGTGATCCAAATTTTACAAACTCCTGAACCAGAA-FL, JAK2-S: 640-IndexTermTTCTCGTCTCCACAGACACAT-P). Direct sequencing of the PCR product was performed by use of BigDye v1.1 chemistry (ABI, Darmstadt, Germany) and a 3130 sequence detection system (ABI).

The first case was represented by a 56-year-old female patient with a 10-year history of nontreated leukocytosis that was interpreted as MPS, not classifiable according to the WHO classification8 (peripheral leukocytes: 17.8 G/l; hemoglobin: 16.3 g/dl; thrombocytes: 494 G/l). The patient was treated with steroids for bronchial asthma for 8 years. Melting-point analysis revealed a pattern different from the typical V617F pattern and beyond the normal range (Figure 1a). Sequencing revealed a heterozygous GAC>GAA exchange at codon 620 leading to an Asp to Glu amino-acid exchange (D620E). It has to be supposed that the function of this mutation is comparable to the V617F mutation resulting in loss of autoinhibition of the JAK2 kinase activity.

Figure 1

Melting curves and results of sequencing analysis. (a) In the case with the GAC>GAA exchange at codon 620 (D620E). (b) In the case with the GAG>GAA exchange at codon 627 (G627G).

The second case was represented by a 50-year-old female patient from Turkish origin who suffered from a still untreated BCR-ABL-negative myeloproliferative syndrome with thrombocytosis, leukocytosis and anemia (leukocytes: 14.2 G/l; hemoglobin: 10.4 g/dl; thrombocytes: 1923 G/l). In this case, sequencing demonstrated a silent heterozygous GAG>GAA base exchange at codon 627 (Glu>Glu; E627E) (Figure 1b). The variant was not reported in the NCBI SNP database as a polymorphism of the JAK2 gene. Unfortunately, analyses from the patient's family were not available. Thus, it remains open whether the respective base exchange represented a rare nucleotide polymorphism or a silent mutation. However, it cannot be completely excluded that such variants enhance JAK2 function, for example, via increased mRNA stability.

To our knowledge, both nucleotide exchanges were not reported yet. We overview 3208 alleles (1604 cases) that were analyzed in our laboratory (data not shown). Although base exchanges other than the V617F seem extremely rare, both cases emphasize the need for diligent analysis of the JAK2 pseudokinase domain to characterize new variants and mutations, to determine their frequency and to improve understanding of the clinical phenotypes associated with these base exchanges. Conventional methods like restriction enzyme digestion or allele specific PCR would overlook these variants as they are restricted to the detection of predefined nucleotide exchanges. The method described here is an alternative to conventional methods using restriction digestion or allele specific PCR to detect such rare variants.


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Correspondence to S Schnittger.

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Schnittger, S., Bacher, U., Kern, W. et al. Report on two novel nucleotide exchanges in the JAK2 pseudokinase domain: D620E and E627E. Leukemia 20, 2195–2197 (2006).

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