Development of High Throughput PCR-Invader Assay to Detect the JAK2 V617F Mutation in Japanese Myeloproliferative Disorders

The classic myeloproliferative disorders (MPD) of polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) are hematopoietic ones characterized by the overproduction of one or more mature myeloid lineages. Although their clonal nature and cytokine independence were recognized a couple of decades ago, their molecular abnormality was not clarified until the identification of the activating JAK2 V617F mutation. Around 95% of patients with PV have the JAK2 V617F mutation. The JAK2 V617F mutation is found in approximately half of ET and IMF patients. ET patients with JAK2 V617F resemble PV patients such as in their increased erythropoiesis and granulopoiesis, and have increased rates of venous thrombosis compared to JAK2 V617F-negative patients. Accurate genotyping of JAK2 is critical for a diagnosis of MPD and can be useful for treatment decisions. However, compared to Western populations, there is limited information available about JAK2 mutation in a Japanese population. This prompted us to analyze JAK2 genotyping utilizing a new method. Although various methods such as direct sequencing, PCR-RFLP, SSCP and DHPLC have been developed for genotyping, none of these provide a rapid, accurate and reproducible system. In this study, a simple assay based on a serial invasive signal amplification reaction assay (PCR-Invader assay) was developed for the detection of JAK2 V617F. The PCR-Invader assay was originally developed for typing SNPs in the human genome. It can detect genotypes in simple 96-well plates without sequence analysis, enzyme digestion or electrophoresis. We analyzed 86 Japanese MPD patients (PV 21, ET 52, IMF 10, others 3). Genomic DNA was extracted from whole blood and the JAK2 V617F mutation then analyzed by the PCR-Invader method. To confirm the accuracy of genotyping by PCR-Invader, the direct sequencing method was also applied to all samples. The percentage of JAK2 V617F mutation-positive patients was PV 81.0%, ET 48.1% and IMF 80.0%. Both white blood cell and platelet counts were higher in V617F-positive PV compared with V617F-negative PV samples (p<0.05). V617F-positive ET samples were accompanied by higher counts of white blood cells and platelets compared with V617F-negative ET ones. However, we did not observe the any relationship between blood cell counts and V617F mutation in IMF; consistent with previous reports. We confirmed that this PCR-Invader method was approximately 20 times more sensitive than direct sequencing. There were no false positive results by PCR-Invader in all 86 samples tested; indicating that PCR-Invader has cost-effectiveness advantages. It has been reported that the activating MPL mutation is observed in some populations of IMF and ET; therefore, we analyzed exon 10 of the MPL genome by direct sequencing. We found that only two V617F-negative ET patients had MPL mutations; W515K and W515-P518delinsKT. There were no MPL mutations in V617F-positive ET patients or all IMF patients. The two ET patients with MPL mutations displayed lower hemoglobin and higher platelets at diagnosis, which is in line with the recently report of the PT-1 study group. In this study we analyzed the JAK2 V617F mutation utilizing the new PCR-Invader assay and undertook MPL mutation by direct sequencing in a Japanese population. Given that some JAK2 inhibitors for MPD patients are now in clinical trials, we expect that this simple PCR-Invader assay and the clinical information about JAK2 and MPL mutations in Japanese MPD patients will be useful to develop clinical trials for JAK2 inhibitors in Japan.