Next-generation sequencing for JAK2 mutation testing: advantages and pitfalls

The JAK2^V617F mutation is part of the major criteria for diagnosis of myeloproliferative neoplasms (MPN). Allele-specific quantitative PCR (qPCR) is the most prevalent method used in laboratories but with the advent of next-generation sequencing (NGS) techniques, we felt necessary to evaluate this approach for JAK2 mutations testing. Among DNA samples from 427 patients analyzed by qPCR and NGS, we found an excellent concordance between both methods when allelic burden was superior to 2% (the detection limit of our NGS assay). Only one sample among 298 was found negative by NGS while allelic burden by qPCR was 3%. Because NGS detection limit is higher, sensitivity was lower as exemplified by 21 samples found negative whereas qPCR measured allelic burdens between 0.1 and 1%. Importantly, quantitative data of samples found positive by both techniques were highly correlated (R² = 0.9477). We also evaluated 40 samples tested for JAK2 exon 12 mutations by HRM. The concordance with NGS was of 100%. Using NGS, the full coding region of JAK2 was analyzed leading to identification of several variants outside of exon 12 and 14 which were previously described or not. Interestingly, we found one somatic mutation (c.1034A>T p.H345L) which induced constitutive activation of the JAK/STAT pathway leading to an increased proliferation of BaF/3 cells with low-dose EPO. This study showed that NGS is a robust method highly correlated to qPCR, although less sensitive, but providing the opportunity to identify other JAK2 variants with potential impact on disease initiation or evolution.