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Development of a Real-Time qPCR Assay for Detection of Common MPL Mutations in Myeloproliferative Neoplasms (MPNS)

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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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Data Availability

All data generated or analyzed during this study are included in this published article.

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Authors and Affiliations

  1. Department of Medical Lab Technology, University of Haripur, Haripur, KP, Pakistan

    Naeem Ullah, Sadiq Noor Khan, Muhammad Umair & Aamer Ali Khattak

  2. Department of Pathology, Northwest Institute of Health Sciences, Peshawar, KP, Pakistan

    Naeem Ullah & Aamir Ali Khan

  3. Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, KP, Pakistan

    Muhammad Umair & Yasar Mehmood Yousafzai

  4. College of Life Sciences and Bioengineering, Beijing University of Technology, Beijing, China

    Aamir Ali Khan & Xinhui Liu

Authors
  1. Naeem Ullah
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  2. Sadiq Noor Khan
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  3. Muhammad Umair
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  4. Aamir Ali Khan
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  5. Xinhui Liu
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  6. Aamer Ali Khattak
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  7. Yasar Mehmood Yousafzai
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Contributions

Naeemullah, Dr. Yasar Mehmood Yousafzai and Dr. Sadiq Noor conceptualized the idea of this study; Naeem Ullah and Muhammad Umair perform the experiments. Dr. Aamir Ali Khan, Dr. Xinhui Liu and Dr. Aamer Ali Khattak write the initial draft of the manuscript. Dr. Sadiq Noor and Dr. Yasar Mehmood Yousafzai validate the data and provide overall supervision.

Corresponding authors

Correspondence to Sadiq Noor Khan, Aamir Ali Khan or Yasar Mehmood Yousafzai.

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Ethics Approval

The study was conducted according to the ethical standards of the Declaration of Helsinki [14], and was approved by the Advanced Studies Research Board (ASRB) of the concerned department.

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Informed consent was obtained from all individual participants included in the study.

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The authors affirm that human research participants provided informed consent for publication.

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All data generated or analyzed during this study are included in this published article.

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The authors declare no competing interests.

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Ullah, N., Khan, S.N., Umair, M. et al. Development of a Real-Time qPCR Assay for Detection of Common MPL Mutations in Myeloproliferative Neoplasms (MPNS). Appl Biochem Biotechnol 194, 5907–5917 (2022). https://doi.org/10.1007/s12010-022-04051-y

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  • DOI: https://doi.org/10.1007/s12010-022-04051-y

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