Rapid and sensitive detection of calreticulin type 1 and 2 mutations by real-time quantitative PCR

BACKGROUND: The majority of patients with JAK2 V617F-negative essential thrombocythemia or primary myelofibrosis harbor mutations involving the calreticulin (CALR) gene. These mutations are located in CALR exon 9 and lead to a frameshift with subsequent alteration of the CALR protein C-terminus. They have emerged as valuable molecular markers for the diagnosis of clonal myeloproliferative diseases. Although a variety of CALR mutations have been described, two mutations, denoted type 1 and type 2, account for around 85 % of cases. The type 1 mutation encompasses a 52 bp deletion and the type 2 mutation a 5 bp TTGTC insertion.

METHODS: This work describes the development and testing of quantitative real-time PCRs (qPCRs) for detecting these two mutations.

RESULTS: The final type 1 CALR qPCR displayed a sensitivity of <0.1 % mutant alleles and the type 2 CALR qPCR had a sensitivity of <0.01 % mutant alleles. Additionally, two new CALR mutations are reported.

CONCLUSION: These sensitive and specific qPCRs should be helpful in establishing the diagnosis and in monitoring minimal residual disease in patients during or after therapy.