Calreticulin mutations in myeloproliferative neoplasms and new methodology for their detection and monitoring

Abstract

The diagnosis of the BCR-ABL-negative myeloproliferative neoplasms (MPN), namely polycythemia vera, essential thombocythemia and primary myelofibrosis has relied significantly on the detection of known causative mutations in the JAK2 or MPL genes, which account for the majority of MPN patients. However, around 30 % of patients with MPN, primarily essential thombocythemia and primary myelofibrosis, lack mutations in these two genes making it difficult to reach a confident diagnosis in these cases. The recent discovery of frameshift mutations in CALR in approximately 70 % of MPN patients lacking the JAK2 and MPL mutations offers a reliable diagnostic marker for the latter group. A review of the current literature, plus unpublished data from our laboratory, shows that 55 different CALR insertion/deletion mutations have been identified so far in MPN patients. Among these 55 variants reported to date, a 52-base pair deletion and a 5-base pair insertion are by far the most prominent representing 50 and 35 %, respectively, of all cases with CALR mutations. In this paper, we describe a high-resolution melting (HRM) analysis and a Taqman® Real-Time PCR (RQ-PCR) assay and we propose a new clinical laboratory diagnostic algorithm for CALR mutation analysis. According to this algorithm, samples can go through front-line screening with HMR or fragment analysis, followed by the newly developed RQ-PCR to both discriminate and quantify the two most common mutations in CALR gene.

CALR mutations screening in wild type JAK2V617F and MPLW515K/L Brazilian myeloproliferative neoplasm patients

“…Since CALR mutations discovery, several studies have been correlated these mutations with clinical data and patient outcome in MPN [14][15][16][17][18][19][20][21][22][23][24]. Interestingly, it was described the presence of CALR mutation in peripheral granulocytes of two PV patients negative for both JAK2 V617F and JAK2 exon12 mutations [25].…”

A novel molecular assay using hybridisation probes and melt curve analysis forCALRexon 9 mutation detection in myeloproliferative neoplasms

“…These two mutations collectively account for 84% of reported CALR mutations (50% and 34%, respectively). The next most frequent mutations in CALR exon 9 are a 46 bp deletion (c.1095_1140del), known as type 3; and a 34 bp deletion (c.1102_1135del), known as type 4, with each of these mutations representing 1.26% of the mutations recently published 2 5 6. Type 5 mutation, a 52 bp deletion (c.1091_1142del), brings the percentage of type 1 to type 5 mutations to 87% of published mutations 5 6…”

“…We report here the development and evaluation of a rapid, easy-to-use assay using LightCycler Hybridisation Probes (HybProbes) and melt curve analysis designed for the detection of type 1–5 CALR mutations, which account for 87% of total mutations discovered to date 6. The hybridisation probe (HybProbe) format used in this study consists of two oligonucleotides (donor and acceptor) that are specifically designed to hybridise to adjacent internal sequences of the amplified fragment during the annealing phase of the PCR reaction.…”

Diversities of Calreticulin Gene Mutations in Macedonian Patients With Essential Thrombocythemia

“…These findings have an important implication for the methodology of CALR defects analyses indicating that the use of methods based on size difference, as those used in our study, or the melting curve analysis should be methods of choice for initial screening of mutations in the CALR gene, as suggested before. 14 The clinical and hematological features of the CALR positive ET patients from our cohort were in line with previously published data. [10][11][12][13] Most notably, CALR mutations were more frequently detected in male patients with higher platelet and lower leucocyte and erythrocyte counts, and lower hemoglobin levels at diagnosis.…”