Development of a Real-Time qPCR Assay for Detection of Common MPL Mutations in Myeloproliferative Neoplasms (MPNS).
Appl Biochem Biotechnol
; 194(12): 5907-5917, 2022 Dec.
Article
em En
| MEDLINE
| ID: mdl-35838887
ABSTRACT
Myeloproliferative neoplasms (MPNs) are blood cell disorders, characterized by overproduction of abnormal cells in bone marrow due to stem cell mutation. The proliferations of blood cell are controlled by many genes particularly MPL gene which encodes thrombopoietin receptor, a hematopoietic growth factor involved in the production and regulation of the platelets and multipotent hematopoietic progenitor cells. Acquired mutations including (W515L and W515K) in this gene have been observed in patients with primary myelofibrosis or essential thrombocythemia lacking JAK2 (V617F) mutations. MPL mutation detection is important for MPNs diagnosis, but due to low frequency of mutant allele burden (< 15%) may be missed by already available common assays such as Sanger sequencing. Furthermore, these techniques are costly, time-consuming, and less sensitive. In present study, we aimed to develop sensitive, less time-consuming, and cost-effective real-time PCR assay for the detection of MPL mutations that is based on TaqMan fluorescent probes. DNA was extracted from blood sample of 128 MPNs patients collected and further analysis was performed on TaqMan RT-PCR. Reference curve was obtained for amplified product of MPL gene containing mutated sequence. The predicted sensitivity level was at least 5% mutant allele burden by our developed assay that is much higher than sequencing output. Out of 128, 2 (1.56%) patients harbored W515L mutation and 1 (0.78%) harbored W515K mutation. It was concluded that TaqMan qRT-PCR assay is an efficient, sensitive, cost-effective, and less time-consuming method capable of detecting MPL mutation in MPNs patients. We suggested that this assay might be helpful in investigating mutant allele load in MPNs patients.
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Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Transtornos Mieloproliferativos
/
Neoplasias
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Appl Biochem Biotechnol
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Paquistão