Mutations in exon 12 of <i>JAK2</i> are mainly found in JAK2 V617F‐negative polycythaemia vera patients

Kouroupi, Eirini; Zoi, Katerina; Parquet, Nathalie; Zoi, Christine; Kiladjian, Jean‐Jacques; Grigoraki, Vassiliki; Vainchenker, William; Lellouche, Franck; Marzac, Christophe; Schlageter, Marie‐Hélène; Dosquet, Christine; Scott, Linda M.; Fenaux, Pierre; Loukopoulos, Dimitris; Chomienne, Christine; Cassinat, Bruno

doi:10.1111/j.1365-2141.2008.07223.x

Cited by 26 publications

(20 citation statements)

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“…Exon 12 mutations on the other hands are extremely variable in sequence [10] and so far restricted to polycythaemia patients. In a recent study, using allele specific PCR, we reported the presence of JAK2 exon 12 mutations in 8 out of 24 PV patients negative for JAK2 V617F mutation, but failed to detect these mutations in patients with idiopathic erythrocytosis [11].…”

Section: Introductionmentioning

confidence: 87%

Interlaboratory Development and Validation of a HRM Method Applied to the Detection of JAK2 Exon 12 Mutations in Polycythemia Vera Patients

et al. 2010

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BackgroundMyeloproliferative 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/FindingsFor 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.ConclusionThe assay we have developed is thus a valid method, adapted to routine detection of JAK2 exon 12 mutations with highly reproducible results.

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Section: Introductionmentioning

confidence: 87%

Interlaboratory Development and Validation of a HRM Method Applied to the Detection of JAK2 Exon 12 Mutations in Polycythemia Vera Patients

et al. 2010

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“…7,11,14,17,20 We based our assay on PCR followed by nucleotide sequencing because a fairly large number of different mutations have been found in this region, and an assay designed to detect only one mutation, such as JAK2 V617F, would not be adequate. To increase the analytical sensitivity of the assay, we used a "clamp" to suppress amplification of the normal sequence.…”

Section: Resultsmentioning

confidence: 99%

“…15 However, several authors have reported patients with a mutation in a small fraction of the isolated DNA, which may not be detected by using standard dideoxy nucleotide sequencing. 7,14,17,20 Our approach was to use an oligo clamp to suppress amplification of the normal sequence and enhance amplification of mutant DNA. This approach has been called clamped PCR or wild-type blocking PCR.…”

Section: Discussionmentioning

confidence: 99%

“…17,19,21,28 Assays based on allelespecific PCR are much more sensitive, but are limited to detecting only known mutations and usually only one mutation per assay. 7,14 Other methods to increase the analytical sensitivity of the assay include using DNA purified from plasma and using RNA as a starting material rather than DNA. 18,29 Another approach to increased analytical sensitivity is to select cells for analysis that are more likely to harbor the mutation.…”

Section: Discussionmentioning

confidence: 99%

“…12,16,18,19 Several groups have reported that the exon 12 mutations are frequently present in only a small fraction of the granulocytes in some patients. 7,11,14,17,20 Thus, the molecular diagnosis of mutations in exon 12 of the JAK2 gene is complicated by heterogeneity of the mutations and low allelic load. Our aim in this study was to establish an assay to detect JAK2 exon 12 mutations with a high degree of analytical sensitivity.…”

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Detection of Exon 12 Mutations in the JAK2 Gene

Laughlin

Moliterno

Stein

et al. 2010

The Journal of Molecular Diagnostics

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JAK2 V617F is the most frequently found somatic mutation in polycythemia vera (PV). Among the cases negative for V617F , a significant fraction have a mutation in exon 12 of the JAK2 gene. Several groups have reported that the exon 12 mutations are present in only a small fraction of the blood cells in some patients. We have developed an assay to detect these mutations with an analytical sensitivity of 0.1% by using a "PCR clamp" to inhibit amplification of the normal sequence and enhance amplification of DNA containing a mutation in the clamp target sequence. The products of this reaction were analyzed by capillary electrophoresis to detect deletions , which are the most frequent type of exon 12 mutations , or by nucleotide sequencing to detect all of the mutations. In a survey of 34 specimens from patients with PV or idiopathic erythrocytosis who did not have a JAK2 V617F mutation , we found four with a mutation in exon 12 , 3 of 10 with PV , and 1 of 24 with idiopathic erythrocytosis. In two cases the mutation was present in a small fraction of the cells and difficult to detect without the use of the clamp. The use of an assay with increased analytical sensitivity enhances the ability to identify patients with mutations in exon 12

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Myeloproliferative and Myelodysplastic/Myeloproliferative Neoplasms and Related Conditions

2019

Bone Marrow Pathology

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Mutations in exon 12 of JAK2 are mainly found in JAK2 V617F‐negative polycythaemia vera patients

Cited by 26 publications

References 10 publications

Interlaboratory Development and Validation of a HRM Method Applied to the Detection of JAK2 Exon 12 Mutations in Polycythemia Vera Patients

Interlaboratory Development and Validation of a HRM Method Applied to the Detection of JAK2 Exon 12 Mutations in Polycythemia Vera Patients

Detection of Exon 12 Mutations in the JAK2 Gene

Myeloproliferative and Myelodysplastic/Myeloproliferative Neoplasms and Related Conditions

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