ISSN: 1300-7777 E-ISSN: 1308-5263
NPM1 mutation analysis in Acute Myeloid Leukemia: Comparison of three techniques Sanger Sequencing, Pyrosequencing, and Real Time PCR. [Turk J Hematol]
Turk J Hematol. Ahead of Print: TJH-66891 | DOI: 10.4274/tjh.2017.0095  

NPM1 mutation analysis in Acute Myeloid Leukemia: Comparison of three techniques Sanger Sequencing, Pyrosequencing, and Real Time PCR.

Dushyant Kumar1, Anurag Mehta2, Manoj Kumar Panigrahi1, Sukanta Nath1, Kandarpa Kumar Saikia1
1Dept Bioengineering & Technology, Gauhati University, Guwahati, India
2Rajiv Gandhi Cancer Institute and research Centre, New Delhi

Objective: Nucleophosmin-1 (NPM1) mutations have prognostic importance in acute myeloid leukemia (AML) patients with intermediate-risk karyotype at diagnosis. Approximately 30% of newly diagnosed cytogenetically normal AML (CN-AML) patients harbor NPM1 mutation in India. In this study we compared the efficiency of three molecular techniques in detecting NPM1 mutation in perepherial blood and bone marrow samples.
Materials and Methods: In a single centre cohort we analyzed 165 CN-AML bone marrow/peripheral blood for NPM1 mutation analysis. Around 30 % CN-AML presented with the NPM1 mutation. For the detection, three methods were compared: Sanger sequencing, Pyrosequencing, and Real-timePCR.
Results: NPM1 exon 12 mutations were observed in 52 (31.51%) of all CN-AML cases. The sensitivity of Sanger sequencing, Pyrosequencing, andReal-time PCR: 80%, 90%, and 95%, whereas specificity was 95%, 100%, 100% respectively. While minimum limit of mutation detection was 20%-30% for Sanger sequencing, 1-5% for Pyrosequencing and 0.1-1% for Real Time PCR.
Conclusion: Sequencing method which is reference method has lowest sensitive and sometimes difficult to interpret. Whereas real-time PCR is a highlysensitive method for mutation detection but limited for specific mutation type, While in our study pyrosequencing emerge as best suitable technique for thedetection of NPM1 mutation detection by Pyr analysis on the basis of its easy of interpretation and less time-consuming processes than sanger sequencing.

Keywords: NPM1, Pyrosequencing, AML, Mutation Analysis




Corresponding Author: Dushyant Kumar, India


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