Prevalence of JAK2V617F, CALR in Philadelphia Positive and Negative Myeloproliferative Neoplasm

1 Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran 2 Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran 3 Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 4 Pediatric Cardiovascular Research Center, Shiraz University of Medical Sciences, Shiraz, Iran 5 Department of Medical Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran 6 Shiraz Paramedical School, Shiraz University of Medical Sciences, Shiraz, Iran GMJ.2021;10:e2127 www.gmj.ir


Introduction
B ased on the World Health Organization (WHO) classification criteria, the classic myeloproliferative neoplasms (MPNs) include chronic myeloid leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) [1]. MPNs are known as disorders of clonal hematopoietic stem cells arising from overproduction of myeloid lineage independent from normal cytokine regulation [2]. Clinical manifestations of MPNs are heterogeneous, ranging from asymptomatic to severe constitutional symptoms and thrombotic disease, or changing to each other or secondary acute myeloid leukemia (AML) [3,4]. Sometimes, the MPNs are divided into Philadelphia chromosome-positive (CML) or Philadelphia chromosome-negative (ET, PV, and PMF) subjects. The understanding of the pathogenesis of Philadelphia negative MPNs was expanded by describing Janus kinase2 (JAK2 V617F) mutation on chromosome 9 in 2005 [5]. JAK2 V617F mutation is detectable in nearly 95% of PV patients and around 50-60% of those with PMF or ET. Also, another somatic mutation in exon 12 of JAK2 is detected in 3-5% of PV patients [6]. Following JAK2 gene mutation, two other recurrent somatic gene mutations, including myeloproliferative leukemia virus (MPL) and recently Calreticulin (CALR) gene mutation, have been detected in patients diagnosed with MPNs, which seems to have an impact on pathogenesis and clinical manifestation of these patients [7]. CALR mutations are deletions or insertions in the terminal exon 9 DNA sequence sub-classified in two subtypes known as type one and two (52bp deletion [p.l 367fs*46; rs1450785140] and 2, 5-bp TTGTC insertion [p. k385fs*47; rs765476509] consequently). These are detected in more than 80% of patients with this gene abnormality [8]. Somatic CALR gene mutations are found in less than 25% of PMF or ET with MPL and JAK2 wild type [9]. The presented study aimed to explore the mutations of CALR and JAK2 genes in an Iranian population of MPN disorder and compare their frequency with worldwide data.

Study Subjects
In this case-control study, 130 MPN patients, according to the WHO criteria, from the Oncohematolgy department at Nemazee Hospital, Shiraz, Iran, from May 2018 to May 2019 were selected. Also, 51 sex-and age-matched control group were selected. The diagnosis of patients was made based on clinical and laboratory evaluation as well as molecular analysis. For all subjects, written informed consent was obtained. The ethical committee of Shiraz University of Medical Sciences, Shiraz, Iran, approved the study (ethical code: IR.SUMS.REC 1397.535).

Molecular Analysis
DNA extraction was done on the peripheral blood cells of all subjects by a commercially available extraction kit (Gene Matrix Quick blood DNA purification kit, EURx, Gdansk, Poland), according to the manufacturing procedure. The DNA sample was stored at -80°C until analysis.
JAK2 Gene Mutational Analysis JAK2 V617F was evaluated based on amplification-refractory mutation system (ARMS)-PCR by designed specific primers. Each PCR mixed contained 5 µl amplicon 2x master mix, 0.8 µl forward and reverse primers, and 0.5 µl common primer, 1 µl DNA template, 1 µl BSA, and 1.7 µl DNase, RNase-free distilled water to complete total volume 10 µl. PCR was carried out using the Thermocycler (Bio-Rad T-100, California, USA) as the following consequence: 5 minutes initial denaturation at 95ºC, then 30 cycles include 30 seconds in 95ºC, 40 seconds in 60ºC and 30 seconds in 72ºC and finally by 72ºC for 5 minutes. Table-1 shows the designed specific primers for each mutation.

PCR Products Evaluation
PCR products for gene mutations were evaluated using 2% agarose gel electrophoresis to identify the CALR gene rs1450785140, rs765476509, and JAK2 V617F gene mutations. The agarose gel electrophoresis result was demonstrated in Figure-1.  Table-2 shows CALR gene mutations frequency in all groups. None of the control groups had any CALR mutation. CALR rs1450785140 was found in four patients with MPNs (one heterozygous and three homozygous states), and 2 out of 4 mutations in this position were detected in Philadelphia chromosome-positive patients (CML cases). CALR rs765476509 was found in five MPNs patients (three heterozygous and two homozygous states); one homozygous state was related to a CML patient. Statistical analysis revealed no any difference among MPN patients based on a diagnosis. It may be related to a small number of CALR mutations in this study. Table-3 shows the frequency of JAK2 gene mutation and its association with CALR mutation in different MPN diagnoses. Figure-2 graphically demonstrates these findings and clearly shows the highest prevalence of JAK2 mutation in PV cases (90% in PV vs. 77.8% in PMF and 51% in ET, P=0.006). In Philadelphia negative cases, most CALR+ mutation was related to ET, but we had no significant statistical difference between ET, PV, and PMF cases due to the small number of CALR mutations, and also no relation to JAK2 mutation could be analyzed. The lowest rate of JAK2 mutation was related to CML cases vs. GMJ.2021;10:e2127 www.gmj.ir  Most CML cases (69%) had negative results for both JAK2 and CALR mutation analysis (Figure-3). Interestingly, 2 of 3 CALR mutations in CML cases were negative for JAK2 mutation (CALR+/JAK2-). A review of the CML clinical profile of patients with CALR mutation did not show any data indicative of other diagnoses, and all three patients were diagnosed based on bone marrow findings and positive BCR/ABL mutation at the initial presentation. None of these three patients developed thrombocytosis after treatment with imatinib. All of them had a good response to imatinib, and in the time of CALR mutation analysis, BCR/ABL had been non-detectable in them.

Discussion
The presentation of JAK2 mutation mainly clarifies the molecular basis and diagnosis of MPNs. Most recently, CALR gene mutations are also presented as another important genetic abnormality, and recurrent mutation is an important factor in the pathogenesis and clinical manifestations of MPNs [13,14]. CALR gene encodes Calreticulin protein, which is located in the lumen of the endoplasmic reticulum that plays a critical role in calcium homeostasis related to apoptosis and cell survival regulation, especially in cancer cells [15]. We evaluated the two most common CALR gene mutations and JAK2 V617F mutation prevalence in Iranian MPNs patients. In all patients, especially in Philadelphia negative cases, the JAK2 gene mutation frequency was high according to other studies [16,17], but the rate of CALR mutation was lower than in other studies. The lower rate of CALR mutations in this study compared to European patients (20-25%) and in Brazilian and Argentinean patients may be related to multiple factors [18][19][20]. Genetic differences of the population may explain the very low CALR mutation rate in the Iranian population. Another explanation may be related to the technique of analysis. The mentioned studies use the whole exon gene analysis for the CALR gene and report a summation of all mutations in this gene. However, our study relied on previous reports that mentioned rs1450785140 and rs765476509 as the most common CALR mutations in patients, presented in 80% of all patients with CALR mutation [10][11][12]21]. Thus, concerning the finding of this study, the CALR mutation in the Iranian population was not as frequent as other ethnicities, or these two types of mutations are not the most common mutations in the Iranian population.
Another study with a whole exon analysis of the CALR gene can further clarify this finding [7,19,20]. However, the low rate of CALR mutation did not allow us to analyze the correlation of CALR mutation with JAK2  [24]. The result of JAK2 mutation analysis was in the same line with those of other studies, clearly showing the JAK2 mutation's role in the diagnosis of PV (90% JAK2+). Even this mutation was statistically more prevalent in PV rather than all other types of MPNs. The rate of JAK2 mutation in CML cases (26.9%) was also in agreement with other studies [25,26].

Conclusion
Our results showed that the JAK2 gene mutation was common among MPNs, especially PV cases. On the other hand, two of the most common mutations of the CALR gene had a very low prevalence in Iranian MPNs but may be seen even in negative JAK2 mutation cases. Further evaluation of the CALR gene by whole exon analysis of CALR is recommended to find the other possible mutation and its dependency on ethnicity and genetic diversity.