CHU BREST, Laboratoire d'Hematologie, Brest, France.
PLoS One. 2010 Jan 26;5(1):e8893. doi: 10.1371/journal.pone.0008893.
Myeloproliferative disorders are characterized by clonal expansion of normal mature blood cells. Acquired mutations giving rise to constitutive activation of the JAK2 tyrosine kinase has been shown to be present in the majority of patients. Since the demonstration that the V617F mutation in the exon 14 of the JAK2 gene is present in about 90% of patients with Polycythemia Vera (PV), the detection of this mutation has become a key tool for the diagnosis of these patients. More recently, additional mutations in the exon 12 of the JAK2 gene have been described in 5 to 10% of the patients with erythrocytosis. According to the updated WHO criteria the presence of these mutations should be looked for in PV patients with no JAK2 V617F mutation. Reliable and accurate methods dedicated to the detection of these highly variable mutations are therefore necessary.
METHODS/FINDINGS: For these reasons we have defined the conditions of a High Resolution DNA Melting curve analysis (HRM) method able to detect JAK2 exon 12 mutations. After having validated that the method was able to detect mutated patients, we have verified that it gave reproducible results in repeated experiments, on DNA extracted from either total blood or purified granulocytes. This HRM assay was further validated using 8 samples bearing different mutant sequences in 4 different laboratories, on 3 different instruments.
The assay we have developed is thus a valid method, adapted to routine detection of JAK2 exon 12 mutations with highly reproducible results.
骨髓增殖性疾病的特征是正常成熟血细胞的克隆性扩张。已经表明，获得性突变导致 JAK2 酪氨酸激酶的组成性激活，存在于大多数患者中。自从证明 JAK2 基因外显子 14 中的 V617F 突变存在于大约 90%的真性红细胞增多症（PV）患者中以来，该突变的检测已成为诊断这些患者的关键工具。最近，在 5%至 10%的红细胞增多症患者中描述了 JAK2 基因外显子 12 中的其他突变。根据最新的 WHO 标准，在没有 JAK2 V617F 突变的 PV 患者中应寻找这些突变。因此，需要可靠且准确的方法来检测这些高度变异的突变。
方法/发现：出于这些原因，我们定义了高分辨率 DNA 熔解曲线分析（HRM）方法的条件，该方法能够检测 JAK2 外显子 12 突变。在验证该方法能够检测到突变患者后，我们验证了它在重复实验中，在从全血或纯化的粒细胞中提取的 DNA 上，都能给出可重复的结果。该 HRM 检测进一步在 4 个不同的实验室、3 种不同的仪器上使用 8 个具有不同突变序列的样本进行了验证。
因此，我们开发的检测方法是一种有效的方法，适用于 JAK2 外显子 12 突变的常规检测，结果具有高度可重复性。
