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A real-time polymerase chain reaction assay for rapid, sensitive, and specific quantification of the JAK2V617F mutation using a locked nucleic acid-modified oligonucleotide

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Abstract
The JAK2V617F mutation has emerged as an essential molecular determinant of myeloproliferative neoplasms (MPNs). The aim of this study was to evaluate the analytical and clinical performances of a real-time PCR (qPCR) assay using a combination of hydrolysis probes and a wild-type blocking oligonucleotide, all containing locked nucleic acid (LNA) bases. Moreover, we validated a procedure for precise quantification of the JAK2V617F allele burden. We used DNA samples from patients suspected to suffer from MPN and dilutions of HEL cells, carrying the mutation, to compare the LNA-qPCR assay to two previously published methods. All assays detected the same 36 JAK2V617F positive patients of 116 suspected MPN diagnostic samples. No amplification of normal donor DNA was observed in the LNA-qPCR, and the assay was able to detect and reproducibly quantify as few as 0.4% of the JAK2V617F allele in wild-type alleles. Quantification of the JAK2V617F allele burden showed similar proportion levels among the different MPN entities as described by other groups. In conclusion, the LNA-qPCR is a rapid, robust, sensitive, and highly specific assay for quantitative JAK2V617F determination that can be easily implemented in clinical molecular diagnostic laboratories. Moreover, precise quantification allows determination of JAK2V617F burden at diagnosis as well as the evaluation of response to JAK2 inhibitors.
Keywords
JAK2-V617F MUTATION, PRIMARY MYELOFIBROSIS, PCR, CHRONIC MYELOPROLIFERATIVE DISORDERS, JAK2 V617F MUTATION, JAK2(V617F) ALLELE BURDEN, MELTING CURVE ANALYSIS, TYROSINE KINASE JAK2, ESSENTIAL THROMBOCYTHEMIA, POLYCYTHEMIA-VERA

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Chicago
Denys, Barbara, Hakim El Housni, Friedel Nollet, Bruno Verhasselt, and Jan Philippé. 2010. “A Real-time Polymerase Chain Reaction Assay for Rapid, Sensitive, and Specific Quantification of the JAK2V617F Mutation Using a Locked Nucleic Acid-modified Oligonucleotide.” Journal of Molecular Diagnostics 12 (4): 512–519.
APA
Denys, Barbara, El Housni, H., Nollet, F., Verhasselt, B., & Philippé, J. (2010). A real-time polymerase chain reaction assay for rapid, sensitive, and specific quantification of the JAK2V617F mutation using a locked nucleic acid-modified oligonucleotide. JOURNAL OF MOLECULAR DIAGNOSTICS, 12(4), 512–519.
Vancouver
1.
Denys B, El Housni H, Nollet F, Verhasselt B, Philippé J. A real-time polymerase chain reaction assay for rapid, sensitive, and specific quantification of the JAK2V617F mutation using a locked nucleic acid-modified oligonucleotide. JOURNAL OF MOLECULAR DIAGNOSTICS. 2010;12(4):512–9.
MLA
Denys, Barbara, Hakim El Housni, Friedel Nollet, et al. “A Real-time Polymerase Chain Reaction Assay for Rapid, Sensitive, and Specific Quantification of the JAK2V617F Mutation Using a Locked Nucleic Acid-modified Oligonucleotide.” JOURNAL OF MOLECULAR DIAGNOSTICS 12.4 (2010): 512–519. Print.
@article{1208690,
  abstract     = {The JAK2V617F mutation has emerged as an essential molecular determinant of myeloproliferative neoplasms (MPNs). The aim of this study was to evaluate the analytical and clinical performances of a real-time PCR (qPCR) assay using a combination of hydrolysis probes and a wild-type blocking oligonucleotide, all containing locked nucleic acid (LNA) bases. Moreover, we validated a procedure for precise quantification of the JAK2V617F allele burden. We used DNA samples from patients suspected to suffer from MPN and dilutions of HEL cells, carrying the mutation, to compare the LNA-qPCR assay to two previously published methods. All assays detected the same 36 JAK2V617F positive patients of 116 suspected MPN diagnostic samples. No amplification of normal donor DNA was observed in the LNA-qPCR, and the assay was able to detect and reproducibly quantify as few as 0.4\% of the JAK2V617F allele in wild-type alleles. Quantification of the JAK2V617F allele burden showed similar proportion levels among the different MPN entities as described by other groups. In conclusion, the LNA-qPCR is a rapid, robust, sensitive, and highly specific assay for quantitative JAK2V617F determination that can be easily implemented in clinical molecular diagnostic laboratories. Moreover, precise quantification allows determination of JAK2V617F burden at diagnosis as well as the evaluation of response to JAK2 inhibitors.},
  author       = {Denys, Barbara and El Housni, Hakim and Nollet, Friedel and Verhasselt, Bruno and Philipp{\'e}, Jan},
  issn         = {1525-1578},
  journal      = {JOURNAL OF MOLECULAR DIAGNOSTICS},
  keyword      = {JAK2-V617F MUTATION,PRIMARY MYELOFIBROSIS,PCR,CHRONIC MYELOPROLIFERATIVE DISORDERS,JAK2 V617F MUTATION,JAK2(V617F) ALLELE BURDEN,MELTING CURVE ANALYSIS,TYROSINE KINASE JAK2,ESSENTIAL THROMBOCYTHEMIA,POLYCYTHEMIA-VERA},
  language     = {eng},
  number       = {4},
  pages        = {512--519},
  title        = {A real-time polymerase chain reaction assay for rapid, sensitive, and specific quantification of the JAK2V617F mutation using a locked nucleic acid-modified oligonucleotide},
  url          = {http://dx.doi.org/10.2353/jmoldx.2010.090137},
  volume       = {12},
  year         = {2010},
}

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