Affiliations 

  • 1 Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia
  • 2 School of Health Sciences, Universiti Sains Malaysia, Kelantan, Malaysia
  • 3 Department of Medicine, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia
  • 4 Faculty of Medical Laboratory Sciences, Department of Haematology, Al-Neelain University, Khartoum, Sudan
  • 5 Diagnostic and Biomedicine, Faculty of Health Sciences, Universiti Sultan Zainal Abidin, Terengganu, Malaysia
Int J Lab Hematol, 2021 Dec;43(6):1451-1457.
PMID: 34125992 DOI: 10.1111/ijlh.13628

Abstract

INTRODUCTION: Calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN) have been reported to be key markers in the molecular diagnosis, particularly in patients lacking JAK2 V617F mutation. In most current reports, CALR mutations were analysed by either allele-specific PCR (AS-PCR), or the more expensive quantitative real-time PCR, pyrosequencing and next-generation sequencing. Hence, we report the use of an alternative method, the conformation sensitive gel electrophoresis (CSGE) for the detection of CALR mutations in BCR-ABL1-negative MPN patients.

METHODS: Forty BCR-ABL1-negative MPN patients' DNA: 19 polycythemia vera (PV), 7 essential thrombocytosis (ET) and 14 primary myelofibrosis (PMF), were screened for CALR mutations by CSGE. PCR primers were designed to amplify sequences spanning between exons 8 and 9 to target the mutation hotspots in CALR. Amplicons displaying abnormal CSGE profiles by electrophoresis were directly sequenced, and results were analysed by BioEdit Sequence Alignment Editor v7.2.6. CSGE results were compared with AS-PCR and confirmed by Sanger sequencing.

RESULTS: CSGE identified 4 types of mutations; 2 PMF patients with either CALR type 1 (c.1099_1150del52) or type 2 (c.1155_1156insTTGTC), 1 ET patient with nucleotide deletion (c.1121delA) and insertion (c.1190insA) and 1 PV patient with p.K368del (c.1102_1104delAAG) and insertion (c.1135insA) inframe mutations. Three patients have an altered KDEL motif at the C-terminal of CALR protein. In comparison, AS-PCR only able to detect two PMF patients with mutations, either type 1 and type 2.

CONCLUSION: CSGE is inexpensive, sensitive and reliable alternative method for the detection of CALR mutations in BCR-ABL1-negative MPN patients.

* Title and MeSH Headings from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.