Rapid, low cost and sensitive detection of Calreticulin mutations by a PCR based amplicon length differentiation assay for diagnosis of myeloproliferative neoplasms

Abstract

Background: Calreticulin (CALR) gene mutations are currently recommended as biomarkers in diagnosis of patients with myeloproliferative neoplasms (MPN) with Jak2 V617F negative phenotype. Our aim was to establish a rapid, low cost and sensitive assay for identification of CALR gene mutations and to validate the diagnostic performance of the established assay in a patient cohort with different clinical MPN phenotypes.

Methods: One hundred five Philadelphia-negative MPN patients, including polycythemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF) were initially screened for JAK2 mutations by amplification-refractory mutation system (ARMS-PCR) methodology and were further subjected to detection of CALR gene mutations by our in-house assay, a PCR based amplicon length differentiation assay (PCR-ALDA). The PCR-ALDA methodology was compared with real time PCR and Sanger sequencing methods. Furthermore, the analytical sensitivity of the assay was established.

Results: PCR - ALDA approach was able to detect and discriminate the pseudo-positive samples containing more than 1% CALR mutant alleles. CALR mutations were not detected in 63 Jak2 V617F positive cases in all three methods. In contrast, amongst 42 Jak2 V617F negative cases, both PCR-ALDA and Sanger sequencing coherently identified 12 CALR mutants compared to 10 CALR mutants detected by real-time PCR method.

Conclusion: PCR-ALDA can be utilized as an easy-to-use, rapid, low cost and sensitive tool in the detection of CALR mutations in Philadelphia-negative MPN patients.

Keywords: CALR mutations; JAK2 V617F; Myeloproliferative neoplasms.

Fig. 1

Study design. All recruited MPN patients were initially screened for BCR-ABL transcripts to differentiate BCR-ABL positive or negative MPN patients using real time PCR. All 105 MPN patients BCR-ABL transcripts negative were subjected to an ARMS-PCR method to screen for the presence or absence of the Jak2 V617F mutation. Irrespective of the presence or absence of wildtype or mutant, all 105 MPN patients were subjected to an in-house developed PCR based protocol (ALDA -amplicon length differentiation assay) for simultaneous detection of CALR type-1 and CALR type-2 mutations. Furthermore the ALDA assay was comparatively evaluated with Sanger sequencing and other available real-time PCR methodologies as described by Zinke et. al [13]

Fig. 2

Distribution of haematological values and Jak2 V617F, CALR mutant alleles in different study population. Panel a: Haematological parameters in: ET, PMF and PV subgroups. Panel b: Age distribution and Jak2 V617F and CALR mutations; PLT: platelets; WBC: white blood cell; RBC: red blood cell; PLT: platelet; Hb: haemoglobin

Fig. 3

Jak2 V617F screening by ARMS-PCR. Panel a with tetra primers: Jak2 V617F mutant allele amplified by primers (Tr-V617F-MT-R/Tr-Jak2-F) resulting in 279 bp product, whereas wild type allele amplified by primer pair (Tr-V617F-WT-F/TR-Jak2-R) resulting in 181 bp product. The amplicon produced by two outer primer pairs (Tr-Jak2-F/TR-Jak2-R) result in 405 bp product (internal control). Panel b Visualization on an agarose gel with dilution series to show resolution of tetra primer PCR product: Upper internal control (405 bp), V617F mutant (279 bp) and the Jak2 wild type (181 bp). Detection is achieved in samples containing up to 0.5% V617F mutant allele

Fig. 4

CALR genotyping and Limit of detection by PCR- ALDA. Dilution series were made by mixing 50% deleted CALR (upper left panel) or inserted CALR (upper right panel) containing DNA samples against wild type DNA. The dilution series were subjected to PCR-ALDA in which only one primer pair is used to unbiasedly amplify wild-type CALR allele and CALR mutant alleles. Subsequently, the reaction mixture was electrophoresed on a 2.5% agarose gel. The 171 bp-CALR-wild-type-allele against the 119 bp (type 1) 52 bp deleted CALR allele or 5 bp TTGTC inserted type − 2 mutation allele or other deleted and inserted alleles. Lower panel- CALR mutations in selected clinical samples: arrows indicate deletions or insertions

Fig. 5

Evaluation of PCR-ALDA compared to Sanger sequencing and real-time PCR. Venn diagram and bar graph illustrating the distribution of Jak2 V617F and CALR mutations among 105 MPN patients. Screening for Jak2 V617F was performed by ARMS-PCR and detection of CALR mutants by PCR-ALDA, real-time PCR (rt-PCR) and by direct Sanger sequencing (SQ)