Development of a multiplex PCR for the rapid detection of clonal eosinophilia

Introduction: Hyper Eosinophilic Syndrome (HES) characterized by persistent eosinophilia. Patients with hypereosinophilic syndrome have a very high incidence of cardiac involvement and carry a poor prognosis without therapy. Fortunately, the results of imatinib therapy in hypereosinophilic syndrome cases with FIP1L1/PDGFRα and ETV6/PDGFRβ fusion genes are very encouraging. Furthermore, A chromosomal aberration involving FGFR1OP2 may be a cause of stem cell myeloproliferative disorder (MPD) that characterized by myeloid hyperplasia, eosinophilia and T-cell or B-cell lymphoblastic lymphoma. For this reason an efficient and reliable methods for rapid detection of these fusion gene is critically important for optimal management of patients. Material and methods: Total RNA was extracted fromleukocytes of a patient with a t(5;12)(q33;p13) positive and EOL-1 and KG1 cell lines using Qiazol. RNA and reverse transcribed into cDNA using random hexamer. The PCR first-step included three primer sets (multiplex). In the nested- PCR, inner primers were designed for each fusion. The PCR internal control (ABL gene) was amplified in all cDNA samples. Result:We have developed a multiplex PCR for the simultaneous detection of FIP1L1/PDGFRα ,ETV6/PDGFRβ and FGFR1OP2–FGFR1 fusion genes in a single amplification reaction. Also nested PCR has developed to monitoring response to therapy. The multiplex PCR and nested-PCR showed a sensitivity of 10-3 and 10-5 respectivly, indicating the assay is suitable for clinical use . Conclusion:Because cytogenetic studies could not find these abnormalities in some patients, molecular detection methods become even more valuable.Molecular confirmation of a PDGFR rearrangement is important for appropriate clinical management and assessment of therapeutic response and monitoring for relapse.

Development of a multiplex PCR for the rapid detection of clonal eosinophilia Keywords:

Hypereosinophilic syndrome , PDGFR rearrangement , FGF R1P2-FGFR1