INTRODUCTION: Whereas the JAK-2V617F allele has been implicated in several of the myeloproliferative disorders, a significant proportion of patients with primary myelofibrosis (MF), or thrombocythemia (ET) are JAK-2V617F negative. One of the factors implicated in these cases is the constitutive activation of JAK-STAT pathway through mutations in the thrombopoietin receptor (MPL) gene. Specifically, mutations at the W515 position are present in approximately 5% of patients with primary myelofibrosis (MF), and in 1% of patients with ET, whereas mutations at the S505 position are common in patients with hereditary thrombocythemia. Mutations of MPL at the S505 and W515 positions have been tentatively linked to severity of myeloproliferative disorders. For this reason, we analyzed MPL sequences of 8700 clinical specimens from JAK-2V617F negative patients suspected to have myeloproliferative disorders.
METHODS: Clinical specimens were obtained and DNA was sequenced from 8700 patients suspected of having myeloproliferative disorders over an 18 month period. Purified DNA was then amplified by PCR on the ABI 9700 Thermalcycler using primers covering a 667 bp region of MPL that contained both the S505 and W515 mutations. The amplified products were subsequently sequenced using on the ABI 3100 or3730 sequencer in both directions for the covered region. These data were analyzed for mutations at S505 and W515 positions.
RESULTS: Of the 8700 patients’ DNA analyzed, 104 had identified mutations at S505, W515 or within in the adjacent region. The majority of individuals with a variant sequence carried the mutations in at the W515 position (n=85/104, 81.7%). The majority of these individuals carried the W515L mutation (n=67, 64.4%), with the remainder carrying W515K (n=11/104, 10.6 %), W515A (n=3, 2.9 %), W515S (n=3, 2.9%) and W515R (n=1, <1%). A smaller number of individuals (n=12/104, 11.5%) had mutations at the S505 position, with all of them carrying the S505N mutation. Lastly, a small subset of individuals (n=7/104, 6.7 %) carried previously identified variants of R514S (n=1, ~1%), R514K (1, %), V507E (n=1, ~1%) and 501A (n=2,%). Of greatest interest was identification of two novel indels at position 515 in two different patients: c.1543_1552delinsAAAA, p.W515_PdelinsKT and c.1543_1559delinsAAAACTGCCAC, p.W515FS.
CONCLUSION: This study examines the MPL mutations in JAK-2V617F negative patients suspected to have myeloproliferative disorders. We identified 104 individuals carrying variant sequences in MPL, predominantly at positions W515 (81.7%) and S505 (11.6%) and in adjacent regions (6.7%) in a population of 8700 individuals. Importantly, we have identified two novel indels at position 515 and further studies needed for the significance.
