Detection of calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN) in Chinese

Abstract

While JAK2 and MPL mutations together are detected in two third of patients with BCR-ABL1-negative MPN, the remaining one third of cases had no genetic marker for diagnosis until the discovery of CALR mutations which fills up the gap. All calreticulin mutations discovered in MPNs were indels in the last exon 9. The commonest mutations are Type 1 and 2 mutations, which represent a specific 52 bp deletion and a 5 bp insertion, respectively. The type of CALR mutation may have prognostic significance in MPN. Sanger sequencing is a powerful method to detect CALR indel mutations, and is regarded as a gold standard. However, this technique is expensive and labor and time-intensive and its analytical sensitivity is only modest. CALR indel mutations can also be examined by fragment length analysis, which uses fluorescein-labeled PCR primers to tag the PCR products and to size them by capillary electrophoresis. It is a relatively simple and sensitive screening method but the exact nature of the indels detected is not known. Amplification-refractory mutation system (ARMS) is a very sensitive method for detection of specific mutations with the use of mutation-specific primers. This allows the amplification of the test DNA only when the target mutation is present. The presence or absence of an amplicon is diagnostic of the presence or absence of the target mutation in the sample. The objectives of this project were to evaluate two molecular techniques, fragment length analysis and ARMS, against the reference method of Sanger sequencing for CALR mutation detection. We have shown that these two techniques are useful for CALR mutation detection. Using these three molecular techniques, we propose a testing algorithm for efficient and cost-effective detection of CALR mutations in JAK2V617F-negative MPN patients in a clinical laboratory setting.