Profile of Calreticulin (CALR) Somatic Mutations in a Cohort of Pakistani Patients with Myeloproliferative Neoplasms
Authors: Ahmed Rifat Zubair , Rashid Munazza , Ahmed Shariq , Nadeem Muhammad , Ahmed Nuzhat , Shamsi Tahir 
Published: October 2019

Abstract
Introduction:
Calreticulin (CALR) is a calcium binding multifunctional protein encoded by the CALR gene, whose job is to bind to mis-folded proteins and prevent them from being exported from the endoplasmic reticulum to the golgi apparatus. Outside the endoplasmic reticulum it is found both intra- and extracellularly on the cell surface and has been implicated in diverse processes including proliferation, apoptosis, phagocytosis and immunogenic cell death.
In December 2013, somatic mutations in exon 9 of CALR were identified as the second most prevalent acquired nucleotide changes in JAK2 mutation negative essential thrombocythosis (ET) and primary myelofibrosis (PMF) patients but not in polycythaemia vera (PV) patients. At that time CALR mutations were found mutually exclusive with JAK2 and MPL. Recently there have been some reports on co-occurence of JAK2 and CALR mutations, not only in ET and PMF but also in some PV cases. Indels in CALR have also been reported in a JAK2 mutation negative PV patient. All CALR mutations are insertions or deletions resulting in a frameshift and cluster in exon 9 which is the last exon of the gene. Thus far, more than 50 different types of mutations in CALR have been reported. Two specific mutations, Type I (52bp deletion) and Type II (5bp insertion), are most prevalent. Overall, these two mutation types are found in more than 80% of patients with mutant CALR. One report found absence of Type II mutations and scatter point mutations in PMF patients of India. In the light of currently available literature, reported from North America (USA), Europe (Cyprus/Italy/Spain), Eastern Europe (Poland), and East Asia (Mainland China/Taiwan China), indel mutations in CALR exon 9 occur with a similar frequency in both ET and PMF patients regardless of ethnic diversity. CALR mutations have been shown to have important diagnostic and prognostic significance in ET and PMF patients and will likely be incorporated into the World Health Organization diagnostic criteria for MPN.
Methods:
Clinical and hematological features were obtained from 51 MPN patients. We studied 10 patients with PV, 17 patients with ET, 12 patients with MF and 9 patients with Undifferentiated MPN. JAK2 V617F mutation was analyzed by DNA tetra-primer amplification refractory mutation system (ARMS-PCR). CALR mutations were identified by bi-directional Sanger sequencing.
Results:
In our cohort, CALR mutations were detected in 7 patients (13.7%; 7/51) of which one patients was positive for JAK2 V617F as well. All those who showed positive results for CALR mutations were ET patients (41.17%; 7/17). Type I mutation was found in 3 patients (42.8%; 3/7) and among them 2 had two new scattered point mutations (c.1081C>G and c.1086C>G) as well, Type II in 2 patients (28.5%; 2/7), and others in 2 patients (28.5%; 2/7). Among those 2 patients with CALR mutations other than Type I or Type II, one had novel CALR mutation pattern of 10bp deletion (c.1130_1139del) whereas other had reported CALR 12bp deletion (c.1214_1225del) profile.
When CALR positive ET patients were statistically compared with JAK2 V617F positive patient, insignificant differences were found in platelet counts (p-value 0.198) and hemoglobin level (p-value 0.380) which might be due to the fact that there was only one JAK2 V617F positive patient in the cohort. No differences were also observed in hemoglobin, white blood cell, platelet counts, hepatomegaly, and splenomegaly between CALR positive and CALR negative patients (p-value >0.05).
Conclusion:
In our cohort of 51 MPN patients CALR mutations were found associated with ET only that is different from other studies worldwide. Prevalence of both scatter point and Type I and Type II CALR mutations are in agreement with worldwide studies but differ from the study conducted on Indian population which shows the need to conduct studies with large sample size in this region to explore the CALR mutation profile in the patients of MPN here.

Disclosures
No relevant conflicts of interest to declare.