ET is a Philadelphia-negative MPN characterized by increasing platelet counts in peripheral blood and clonal proliferation of the megakaryocytic lineage in BM (24, 25). Although this neoplasm is associated with longer overall survival, patients' life expectancy may be reduced due to the occurrence of thrombosis, hemorrhage, and the risk of hematological malignancies transformation (such as secondary myelofibrosis or acute myeloid leukemia) compared to the general population. (23, 26). The overall risk of arterial or venous thrombosis in these patients is estimated at 1–3% per patient-year; Therefore, thrombosis is a life-threatening risk factor in ET patients (27). Numerous acquired factors (advanced age, prior history of thrombosis, and vascular risk factors), genetic risk factors, and some changes in hematological parameters are involved in the pathogenesis of thrombosis in these patients (28). The presence of driver genetic mutations (such as JAK2 V617F) is engaged in ET pathogenesis and is considered one of the culprits of thrombotic attacks (29). On the other hand, some studies have revealed that leukocytosis (leukocyte count > 11 × 10 9/L) or abnormal platelet counts (platelet count ≥ 450×10 9/L) were strongly associated with thrombosis/thrombohemorrhagic complications (5, 30, 31). Many platelet defects, including abnormal platelet aggregation, decreased functionality, acquired storage pool disease, and reduced levels of membrane adhesion molecules (i.e., GP Ib, IIb-IIIa, IV, and VI), have been identified in ET patients to increase the risk of thrombosis (28, 32). Recent studies have also revealed that PEAR1 protein as a transmembrane receptor (a type 1 receptor tyrosine kinase from the Epidermal Growth Factor family) could alter megakaryocytopoiesis through the PI3K/PTEN pathway (33). This protein can also enhance platelet aggregation by increasing the stability of αIIbβ3 on the platelet surface (34). This transmembrane receptor is present on the surface of resting platelets as well as α-granules in platelets. FcɛRIα (IgE receptor) acts as a ligand for PEAR1, which their connection stimulates platelet aggregation and degranulation (12, 35, 36). Out of five intracellular domains of PEAR1, the one that is rich in proline (EMI domain) is responsible for platelet-platelet adhesion (34). The presence of PEAR1 polymorphisms during MK differentiation can lead to unexpected events in the size, platelet aggregation, and the number of mature platelets. Several polymorphisms have been identified in the PEAR1 gene, such as rs41299597, rs3737224, rs41273215, rs82242, rs11264579, rs12041331, and rs12566888, which increase PEAR1 expression and significantly affect platelet aggregation (37). Genetic researchers have discovered that more than 15% of platelet dysfunction cases are related to rs12041331variant. Other studies have also revealed that rs12041331 and rs12566888 variants account for over 1% of platelet phenotypic variation (15, 38, 39). These two PEAR1 variants (rs12041331 and rs12566888) are located far from each other in the PEAR1 gene, but the GG allele in rs12041331 is closely related to the TT allele in rs12566888 and change the platelet activity with a similar mechanism (16, 40). However, the G allele in the rs12041331 variant is more strongly associated with increased platelet aggregation than the T allele in the rs12566888 variant and other PEAR1 variants (16, 41).
For the first time in this study, we surveyed 105 ET patients (and the same number of healthy donors as a control group) to assess the prevalence of rs12041331 and rs12566888 variants in these patients. Additionally, we evaluated the relationship between these variants and hematological parameters (platelet count, WBC counts, and Hb levels), hematological symptoms (thrombosis and hemorrhage), as well as their convergence with ET-related mutations. This study's innovative idea stems from the common presence of thrombosis/platelet aggregation in both ET patients and patients carrying PEAR1 variants in platelet-related disorders, which has not been investigated before. We first identified ET patients based on a 2016 WHO classification through hematological/clinical findings and cytogenetic analysis (Fig. 2). Furthermore, the BM of all ET patients was examined to report the phenotype and number of megakaryocytes (Fig. 1). In the present study, ARMS-PCR was used to track rs12041331 and rs12566888 variant (then confirmed by DNA sequencing), and ET-related mutations were identified with the Allele-specific PCR and Sanger sequencing (Figs. 3 and 4). As expected, the frequency of JAK2 mutation in ET patients was 56.1%. The incidence of CALR and MPL mutations in these patients was 17.5% and 3.5%, respectively. The prevalence of JAK2 (f:m 1.02) and CALR (f:m 1.10) mutations was approximately the same among men and women, while all MPL+ patients were female. Meanwhile, the prevalence of the rs12041331 variant (43.9%) was more prominent than rs12566888 (38.5%) in these patients. The incidence of ET-related mutations was almost similar in both rs12041331 (JAK2: 55.6% > CALR: 30% > MPL: 0.0%) and rs12566888 variant (JAK2: 43.8% > CALR: 30% > MPL: 0.0%). Despite the relatively high prevalence of JAK2 mutation in rs12041331+ patients, no significant relationship was found, but a meaningful relationship was observed between CALR mutation and the presence of rs12041331 variant in these patients (P-Value: 0.03, df: 2). It is thought that the relationship between ET-related mutations and the presence of PEAR1 variants to be more significant in a larger statistical population; So, further research is required in this regard. Evaluation of the effects of PEAR1 variants on platelet count of ET patients revealed that the presence of rs12041331 is significantly associated with an increase in platelet count (P-Value: 0.02, df: 2). Considering the prominent influences of PEAR1 variants on the platelet function/activation and stimulating platelet aggregation in diseases related to platelet defects (such as SPS, DVT, KD, CADs, DVT, etc.), discovering the correlation between the presence of rs12041331 variant and increasing the platelet count in ET patients can be a cornerstone for new research on the effects of PEAR1 variants on platelet function/activation in ET or other platelet-related diseases. In contrast, no significant association was found between PEAR1 variants, WBC counts, and Hb levels. Follow-up of the prevalence of thrombotic events in ET patients indicated that thrombosis occurred in 57.1% of patients with homozygous rs12041331 and 43.8% of patients with rs12566888, but no statistically significant relationship was found between them. In parallel with the evaluation of PEAR1 variants, the results derived from the relationship between ET-related mutations and hematological parameters/symptoms in these patients underscored other recent studies' results. Recent studies on ET patients have proven that platelet counts in JAK2 + patients are lower than in CALR + patients, while WBC counts, Hb levels, and thrombosis incidence in JAK2 + patients is higher compared to CALR + patients (42). This study confirms the relationship between ET-related mutations with hematological parameters/symptoms as in previous studies (43, 44). Results of the present study, just like recent research, reveal that the mean platelet count in patients with JAK2 mutation (790.11 ×10 9/L ± 265.35 SD) is lower than patients with CALR mutation (934.45 ×10 9/L ± 275.09 SD), and the presence of JAK2 mutation is significantly associated with a decreased platelet count in these patients (P-Value: 0.04, Sig: 0.928, df: 103). Besides, WBC counts, Hb levels, and the prevalence of thrombosis was higher in JAK2 + patients compared to CALR + patients, which also confirms the results of recent studies (43). Since the presence of JAK2 mutation is associated with increased WBC counts and a high prevalence of thrombosis, this mutation should be considered more than other ET-related mutations in the diagnosis, prognosis, and management of ET patients.
In recent years, researchers have focused on PEAR1 variants in various diseases, including SPS, CVDs, ACS, CADs, KD, and DVT (45–50). Some other studies have also investigated signaling, functional mechanisms, and protein expression of the PEAR1 gene. Faraday et al. (2011) sequenced the PEAR1 gene and discovered that the rs12041331 variant accounts for ≤ 15% of phenotypic variation in platelet function (16). Lei Pi et al. (2018) examined rs12566888 and rs12041331 variants for the risk of developing coronary artery aneurysm (CAA) in KD. Their results revealed a significant link between rs12041331 variant and CAA occurrence in KD, confirming the association between PEAR1 SNPs and cardiovascular disease, similar to previous studies (49). Keramati et al. (2018), who studied the effect of PEAR1 variants on platelet activation and atherosclerotic plaques in CAD patients, reported that the signaling of rs12566888 was not independent of rs12041331variant. Conversely, their findings emphasized that in the presence of rs12041331 variant, the effect of the rs12566888 variant was less pronounced and that the rs12041331 variant could maintain its impact on platelet activation phenotype independently and more strongly (51). In the first large platelet function genome-wide association study (GWAS), John et al. (2010) demonstrated that two correlated intronic PEAR1 variants (rs12041331 and rs12566888) are linked with increased platelet count, ADP- and epinephrine associated platelet aggregation and platelet adhesion (38). In this study, the rs12041331 variant was associated with an increase in platelet count, which, like previous studies, indicates that this variant could be a threat to stimulate platelet aggregation and thrombosis. On the other hand, some further genetic investigations, including targeted re-sequencing, exome sequencing, and targeted SNP genotype studies, have failed to find a convincing association between PEAR1 variants and platelet activation traits, concluded that rs12041331 variant is the most likely a platelet functional variant (16, 52, 53). Criel et al. and Vandenbriele et al. in 2016 demonstrated that while the PEAR1 variants in the mice models reflected features of platelet activation, platelet function in humans was less affected by these variants (54, 55). Therefore, due to the novelty of the PEAR1 gene, there is still no consensus regarding its effect on platelets functional in many diseases and malignancies. We designed this study in ET patients before large-scale quantitative researches to evaluate the prevalence of PEAR1 variants, their effect on the hematological parameters/symptoms, and their relationship to ET-related mutations. This experiment was also performed to avoid wasting time and money on an inadequately designed project, and it can be a cornerstone for more comprehensive research. Suppose we consider the rs12041331 variant as a co-factor for thrombosis progression (along with JAK2 mutation) and increasing platelet counts. In that case, it may be argued that the presence of this variant could be a risk factor for thrombocythemia and platelet defects in ET patients. However, more extensive research is needed to substantiate such claims. Finally, we point out some of the limitations of this study; First, in this research, we only focused on two of the potentially functional PEAR1 SNPs, which may overlook other important PEAR1 variants. Second, influences of other external factors that may affect platelet counts, including lifestyle behaviors and therapy methods, have not been considered. Third, the small statistical population is another limitation of this study, and we recommend that further research be conducted with a larger target community.