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.