Characterization of FKBP51 as a gene overexpressed in PMF megakaryocytes.

(A) PCR differential screening. mRNA from normal or PMF spontaneously grown megakaryocytes were reverse transcribed and amplified by PCR using AP76 and H-T11-A primers (Genhunter). Amplified cDNAs were run side by side on a sequencing gel. The arrow indicates b49 band only amplified in myelofibrosis samples. (B) Alignment of b49 (288 bp) and FKBP51. Alignment was performed using LFASTAn-LALIGNn program from Infobiogen (Villejuif, France). Sequence homology was 99.6%; b49 alignment was located at the 3′end of the cDNA (base 1445-1733). The 2 18mer initially used to confirm the differential expression in semiquantitative RT-PCR are underlined. (C) Semiquantitative RT-PCR from normal (C) and pathologic (P) MKs. Internal differential display primers were designed from b49 and 27, 29, 32, and 35 cycles were performed to amplify actin and GPIIb. Similar results were obtained with 2 other pathologic samples. (D,E) Real-time quantitative RT-PCR. Real-time quantitative RT-PCR was performed with RNA extracted from spontaneously growing MKs from 8 patients (P1-P8) and MKs grown in presence of SCF and TPO from 4 controls (N1-N4). MKs were immunomagnetically purified; purity of recovered cells was determined by flow cytometry and was over 90%. Ratio of FKBP51 expression on GPIIb mRNA expression showed a 2- to 10-fold increase in FKBP51 expression except in one patient (P6). P6 presented an acute transformation of the disease. The same results were obtained with a ratio between FKBP51 expression and 18S in 6 patients (P1-P6; panel E).