title: A highly specific q-RT-PCR assay to address the relevance of the JAK2WT and JAK2V617F expression levels and control genes in Ph-negative myeloproliferative neoplasms.
abstract: In Ph- myeloproliferative neoplasms, the quantification of the JAK2V617F transcripts may provide some advantages over the DNA allele burden determination. We developed a q-RT-PCR to assess the JAK2WT and JAK2V617F mRNA expression in 105 cases (23 donors, 13 secondary polycythemia, 22 polycythemia vera (PV), 38 essential thrombocythemia (ET), and 9 primary myelofibrosis (PMF)). Compared with the standard allele-specific oligonucleotide (ASO)-PCR technique, our assay showed a 100 % concordance rate detecting the JAK2V617F mutation in 22/22 PV (100 %), 29/38 (76.3 %) ET, and 5/9 (55.5 %) PMF cases, respectively. The sensitivity of the assay was 0.01 %. Comparing DNA and RNA samples, we found that the JAK2V617F mutational ratios were significantly higher at the RNA level both in PV (p = 0.005) and ET (p = 0.001) samples. In PV patients, JAK2WT expression levels positively correlated with the platelets (PLTs) (p = 0.003) whereas a trend to negative correlation was observed with the Hb levels (p = 0.051). JAK2V617F-positive cases showed the lowest JAK2WT and ABL1 mRNA expression levels. In all the samples, the expression pattern of beta-glucoronidase (GUSB) was more homogeneous than that of ABL1 or β2 microglobulin (B2M). Using GUSB as normalizator gene, a significant increase of the JAK2V617F mRNA levels was seen in two ET patients at time of progression to PV. In conclusion, the proposed q-RT-PCR is a sensitive and accurate method to quantify the JAK2 mutational status that can also show clinical correlations suggesting the impact of the residual amount of the JAK2WT allele on the Ph- MPN disease phenotype. Our observations also preclude the use of ABL1 as a housekeeping gene for these neoplasms.
authors.affiliation: Department of Medico-Surgical Sciences and Biotechnologies, University of Rome "Sapienza" Polo Pontino, , Latina, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Fantasia
authors.forename: Emma Nora
authors.initials: EN
authors.lastname: Di Capua
authors.forename: Natalia
authors.initials: N
authors.lastname: Cenfra
authors.forename: Gloria
authors.initials: G
authors.lastname: Pessina
authors.forename: Sergio
authors.initials: S
authors.lastname: Mecarocci
authors.forename: Angela
authors.initials: A
authors.lastname: Rago
authors.forename: Ettore
authors.initials: E
authors.lastname: Cotroneo
authors.forename: Anna
authors.initials: A
authors.lastname: Busanello
authors.forename: Francesco
authors.initials: F
authors.lastname: Equitani
authors.forename: Francesco
authors.initials: F
authors.lastname: Lo-Coco
authors.forename: Clara
authors.initials: C
authors.lastname: Nervi
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Cimino
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D053614:Janus Kinase 2 / Q000096:biosynthesis / Q000235:genetics*; D020014:K562 Cells; D054438:Leukemia, Myeloid, Chronic, Atypical, BCR-ABL Negative / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009196:Myeloproliferative Disorders / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*
chemical_list: C507924:JAK2 protein, human; D053614:Janus Kinase 2