Introduction

The JAK2V617F mutation is observed in more than 95% of patients with Polycythemia Vera (PV), and 50–60% of patients with Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and Refractory Anaemia with ringed Sideroblasts with marked Thrombocytosis (RARS-T). As a consequence, for JAK2V617F-negative cases it is worth looking for other mutations in the JAK2 and MPL genes as they are an important diagnostic aid for the myeloproliferative neoplasms (MPN). For example, half of JAK2V617F-negative PV patients carry mutations in the exon 12 of JAK2. To date, 40 different JAK2 exon 12 mutants have been reported. All are located between aa536 and aa547 and include deletions, insertions, and duplications. In the same way, nearly 5% and 10% of JAK2V617F-negative ET and PMF carry mutations in exon 10 of MPL, mainly affecting codons S505 and W515. Techniques such as allele-specific polymerase chain reaction (AS-PCR) are inappropriate for screening and sequencing, including Next Generation Sequencing (NGS), is time consuming as a first screening. High-resolution melting (HRM) curve analysis allows a rapid and whole exon mutation scanning approach associated with excellent sensitivity. The goal of our study was to evaluate the relevance of HRM in JAK2V617F-negative MPN and RARS-T in 3 French hospitals.

Methods

HRM is based on the analysis of fusion curves of a short amplification product of PCR. This procedure, which derives PCR in real time and uses a fluorescent agent intercalated DNA, makes it possible to obtain a good resolution and to detect different types of mutations. The analyses were carried out on DNA from purified blood granulocytes. Diagnosis of PV, ET, PMF and RARS-T was established according to the 2008 WHO criteria. All patients were tested for their JAK2V617F status. To evaluate the efficiency of HRM in a routine diagnostic setting, we investigated 1997 patients with suspicion for a MPN from 3 French university hospitals. Samples with abnormal HRM curves were sequenced in order to confirm and characterize the mutations.

Results

For 37/881 (4.2%) JAK2V617F-negative IE and PV patients, the HRM curve raised suspicion of the presence of a mutation in JAK2 exon 12, all confirmed by sequencing: the most frequent mutations were H538Q K539L (n=6), R541 E543 delins K (n=6), N542 E543 del (n=5), F537–I546 dup 10_F547L (n=4). For 2 additional PV cases, an abnormal HRM curve was obtained for JAK2 exon 14. Sequencing analysis revealed a V617F mutation with additional mutation of base 1831 (1831T>G), changing leucine 611 for a valine (L611V). For the second PV patient, a silent mutation corresponding to a substitution of a cytosine by a thymine in position 1848 of exon 14 of JAK2 was observed (C616), also associated with a V617F mutation. In both cases the JAK2V617F mutation had not been detected because the primers of the JAK2V617F AS-qPCR assay covered the sequence coding for L611 and C616. In addition, mutations in JAK2 exons 13 and 15 were tested on 33 PV-suspected samples: only one PV with a JAK2 exon 15 mutation was noted. Regarding the detection of mutations in exon 10 of MPL, an abnormal HRM curve was noted for 59/1116 (5.3%) MPN suspected. The mutations found were W515L (31 ET, 4 PMF), W515K (8 ET, 3 PMF), W515A (2 ET), W515R (1 ET, 1 PMF), W515S (1 ET), S505N (4 ET, 2 PMF, 1 RARS-T), V501A (1 ET).

Conclusion

In ET and PMF, the HRM approach offers the advantage of quickly and reliably assessing patients for W515 and S505 mutations. In JAK2V617F-negative PV, due to the large number of possible mutations, HRM is a highly potent screening technique and allows a rapid scan of the whole exon 12, leading secondarily to sequence only a small number of HRM positive cases. Moreover, in 2 PV, HRM analysis of JAK2 exon 14 allowed to detect a V617F mutation hidden by a second mutation that inhibited primer hybridization and subsequent DNA amplification in the routine AS-qPCR assay. In summary, HRM is a highly relevant technique, cheaper than NGS, for routine molecular diagnosis of JAK2V617F-negative MPN, which typically carry a large variety of mutations, expressed at low levels.

Figure 1
Figure 1. Examples of HRM curves of PV JAK2 exon 12 mutations
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Examples of HRM curves of PV JAK2 exon 12 mutations

Figure 1
Figure 1. Examples of HRM curves of PV JAK2 exon 12 mutations
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Examples of HRM curves of PV JAK2 exon 12 mutations

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Disclosures:

No relevant conflicts of interest to declare.

Topics:
high-resolution melt curve analysis, myeloproliferative disease, refractory anemia with sideroblasts, polymerase chain reaction, massively-parallel genome sequencing, screening, dna, crossbreeding, cytosine, endothelin-1

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2013 by The American Society of Hematology
2013
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