PEAR1 Genetic Variants in Essential Thrombocythemia: A New Study of The Prevalence and Association of PEAR1 Variants with Hematological Parameters and ET Mutations

Objective: Essential thrombocythemia (ET) is a type of myeloproliferative neoplasm characterized by the expansion of the megakaryocytic/platelet line. Given the undeniable role of genetic variations in the pathogenesis of ET, as well as the proven effects of PEAR1 SNPs on platelet function, the innovative purpose of this study is to investigate the prevalence of PEAR1 variants (rs12041331 and rs12566888) and their relationship to hematological parameters and ET-related mutations. Materials and Methods: We studied 105 ET patients and analyzed ET patients' mutational proles, including JAK2 V617F mutation (detected by Allele-specic PCR), CALR, and MPL mutations (both through PCR amplication). Two SNPs of the PEAR1 gene were assessed through ARMS-PCR, and the Sanger method was used for the validation of ARMS-PCR amplication. Results: The prevalence of rs12041331 and rs12566888 in ET patients were 43.9% and 38.5%, respectively, and rs12041331 was signicantly associated with increased platelet counts (P-Value: 0.02). A signicant relationship was also found between the rs12041331 and CALR mutation (P-Value: 0.03). Platelet count was higher in CALR + patients (934.45 ×10 9 /L ± 265.35 SD) than in JAK2 + patients (790.11 ×10 9 /L ± 265.35 SD). Conversely, other hematological parameters and thrombosis were higher in JAK2 + patients than the CALR + patients. Conclusions: Our ndings reinforce the idea that rs12041331 and rs12566888 may be associated with ET, and rs12041331 is signicantly associated with increased platelet count. Besides, the prevalence of ET-related mutations in patients with rs12041331 and rs12566888 was almost similar; however, only CALR mutation had a signicant relationship with rs12041331. Two intronic PEAR1 variants with platelet hyper-reactivity selected according to and the sequence was retrieved from the NCBI Because these polymorphisms are due to the single nucleotide changes in the alleles (rs12566888; G/T and rs12041331; G/A), the ARMS-PCR technique was considered a reliable method to detect these point mutations. This technique can also differentiate between heterozygous and homozygous individuals in terms of a single gene locus. For this purpose, two parallel reactions were performed in two separate tubes. In both of these reactions, a similar DNA sample was used. The rst reaction (in tube 1) contained primers specic for normal DNA (normal forward/reverse primers) that could not replicate the mutated DNA at the gene locus. Conversely, the second reaction (in tube 2) contained a mutant allele-specic primer and normal primer (normal forward/mutant reverse), so it could not amplify normal DNA. We used three types of primers; Normal forward primer was constant, and its complementary sequence was present in both reactions. This primer was designed for regions of the gene that often lack mutation and serve as internal controls. The normal band was observed in all tubes containing normal primers. The normal tube (containing regular primers) is a kind of biological control in this study to verify ARMS-PCR results' accuracy. The other two types of primers are different at the 3 'end. The rst (normal reverse primer) is specic to the natural DNA sequence, and the second (mutant reverse primer) is specic to the mutated nucleotide. ARMS primers were designed by Batch Primer 3 software, which is freely available online at (http://probes.pw.usda.gov/cgi-bin/batchprimer3/batchprimer3.cgi). The mismatch was deliberately added near the 3 'end of the primer (at the terminal and proximal nucleotides) to prevent


Introduction
Essential thrombocythemia (ET) is a type of acquired stem cell-derived clonal disease associated with the expansion of megakaryocytic (MK)/platelet line. ET has been categorized as a myeloproliferative neoplasm (MPN) by Dameshech since 1954 (1,2). This neoplasm's primary cause is the hyperproliferation of hematopoietic cells (HSCs) due to genetic driver mutations potentiating the myeloproliferative polymorphisms in the PEAR1 gene to investigate their association with hematological parameters, including platelet count, white blood cells (WBCs) counts, hemoglobin (Hb), and hematological symptoms such as thrombotic events and hemorrhage as well as their prevalence in ET patients. Additionally, we evaluated the prevalence of these polymorphisms in ET and their convergence with ET-related mutations.
The innovative goals and challenging hypotheses of this study are classi ed below; Is the prevalence of rs12041331 and rs12566888 variants in ET patients signi cant?
Is there a meaningful relationship between rs12041331/rs12566888 variants and platelet count, WBCs counts, and Hb levels?
Is there a signi cant association between rs12041331/rs12566888 variants and hematological symptoms such as thrombotic events and hemorrhage in ET patients?
Is there any signi cant relationship between the presence of rs12041331/rs12566888 variants and the occurrence of ET-related mutations?
Investigating the effects of ET-related mutations on hematological ndings and comparing these effects with rs12041331/rs12566888 variants?

Study subjects and Specimen collection
A total of 105 patients (50.0 % female and 42.1 % male) with a con rmed diagnosis of ET and Philadelphia chromosome-negative (Ph − ) according to 2016 World Health Organization (WHO) criteria were entered in the present study between November 2018 and March 2020 that referred to the Ahvaz Baghaie 2 hospital ( Table 1). The laboratory hematologic ndings of ET patients (including platelet count, WBC count, hemoglobin) along with clinical symptoms (thrombotic events and hemorrhage) and cytogenetic tests were under follow-up in Jundishapur University Department of Clinical Laboratory Sciences and recorded at Noor Genetics Lab of Ahvaz. The inclusion criteria in this study were considered 2016 WHO classi cation for ET (17,18). Exclusion criteria were as follows: erythrocytosis, Philadelphia chromosome-positive (Ph+), leucoerythroblastic blood picture, morphological abnormalities compatible with PMF, PV, myelodysplastic syndromes (MDS), or other myeloid neoplasms, and evidence for reactive thrombocytosis. This study is based on the approval of the Medical Ethics Committee of Jundishapur Ahvaz University (IR.AJUMS.REC.1398.571). After obtaining written consent from all patients, 3 ccs of venous blood for analysis of ET-related mutations and PEAR1 SNPs (rs12041331 / rs12566888) was taken from them and collected into dipotassium ethylenediaminetetraacetic acid (K2EDTA)-anticoagulated tubes. Total DNA was extracted from venous blood specimens by using the QIAamp DNA Mini Kit (Germany) according to the manufacturer's instructions and preserved at − 70° C. Thermo NanoDrop One Microvolume ultraviolet-visible spectrophotometer (Thermo Fisher, USA) was used to determine DNA concentration and quality with a concentration of 100-200 ng/µl and 1.8-2.0 ratio in 260/280 nm (260/280 ratio). The samples with a concentration of less than 20 ng/µl were selected for this study. Besides, peripheral blood samples collected from 105 healthy donors were also used as controls in this study.

ET diagnosis
In this study, patients were evaluated in terms of complete blood count, BM examination, and genetic testing. The criteria for ET diagnosis were according to the 2016 WHO criteria (Fig. 1). Platelet counts in all patients were ≥ 450×10 9 /L, and the mean platelet count was 835.33 ×10 9 /L ± 284.56 SD. Hematologists reviewed BM aspirates; an increased proliferation of megakaryocytic cell line along with increased enlarged/maturated megakaryocytes was signi cant evidence in patients' aspirates ( Fig. 1). Because most MPNs have overlap with each other or with other diseases, it is essential to rule out other causes of thrombosis, such as reactive thrombocytosis (in in ammation and iron de ciency) before ET is diagnosed (5). For this purpose, thrombosis removal by iron replacement or the resolution of in ammation led to differentiate reactive thrombosis from ET. Given that ET is a type of Philadelphia-negative MPN, it was initially necessary to prove the Philadelphia chromosome's absence in all patients. Since sensitive and high-speed methods are needed to detect the BCR-ABL fusion gene, a multiplex reverse transcriptionquantitative real-time PCR (Multiplex RT-qPCR) was performed in Ahvaz Noor Genetics Lab based on the approach published by Burmeister T. and Tong Y. et al. (19,20). The genetic testing pattern (JAK2, CALR, and MPL mutations) was applied for all patients to diagnose ET based on 2016 WHO protocol, as shown in Fig. 2. Allele-speci c polymerase chain reaction (PCR) was used to detect JAK2 V617F mutation (21).
JAK2 V617F-negative patients were sequenced for CALR or MPL mutations through PCR ampli cation and Sanger sequencing (22). Allele-speci c PCR for the detection of JAK2 V617F mutation (with a sensitivity de ned as 0.5-2%) was performed with 5 µl of genomic DNA in the reaction mixtures contained 12.5 µL 2× TaqMan PCR Master Mix, TaqMan probe, primers (1 µL common reverse primer, and 0.5 µL each of forward primers), and distilled water. The primers sequences and probe used for PCR ampli cation of JAK2 V617F were as follows: Forward primer 1, 5′-AGCATTTGGTTTTAAATTATGGAGTATATT-3′, Forward primer 2, 5′-ATCTATAGTCATGCTGAAAGTAGGAGAAAG-3′, Reverse primer, 5′-CTGAATAGTCCTACAGTGTTTTCAGTTTCA-3′, and probe, (6FAM AATTATGGAGTATGTTTCTGMGBFNQ). Forward primer 1 (Forward wild-type-speci c primer) only in the presence of a JAK2 V617F mutation generates 203 bp PCR product, while Forward primer 2 (Forward mutant-speci c primer) anneals to both normal and mutant alleles and produces 364 bp PCR product, so it was used as an internal control. The PCR conditions included an initial denaturation of 5 minutes at 95°C, followed by 35 cycles of 30 seconds at 95°C (denaturing), 30 seconds at 58°C (annealing), 45 seconds at 72°C (elongation); and nal extension of 72°C for 5 min.
The following forward and reverse primers were used for PCR ampli cation of CALR and MPL genes; CALR forward primer, 5′-CAT TCA TCC TCC AGG TCA AG-3′; CALR reverse primer, 5′-AGG GGA ACA AAA CCA AAA TC-3′; MPL forward primer, 5′-TGG GCC GAA GTC TGA CCC TTT-3′; and MPL reverse primer, 5′-ACA GAG CGA ACC AAG AAT GCC TGT-3′. PCR was performed as follows; 25-100 ng DNA in 100 µL PCR solution including, 10× MG Taq-HF buffer (10 µL), 2 mmol/L MG dNTPs mixture (10 µL), MG Taq-HF polymerase (1 µL), forward/reverse primers (0.2 µmol/L of each), and distilled water. PCR reactions were performed as follows; initial denaturation at 95 C for 5 min followed by 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, and nal extension of 72°C for 5 min. 2% agarose gel in 1X TBE buffer was used for electrophoresis, which was run for 50 min in 110 voltage (PAYA PAZHOOHESH, Iran). Then, the electrophoresis gel was visualized through UV light in a gel documentation system. Sanger sequencing was carried out via ABI-3130 XL (USA) using the above-described Forward/Reverse primers, and UGENE software was used for visualization of the sequences data (Fig. 5). The resultant sequences of CALR and MPL genes were compared with sequences on the GenBank website (http://www.ncbi.nlm.nih.gov/sites/Entrez). In every run (to detect JAK2, CALR, or MPL mutations) and ampli cation of the DNA templates, reactions with negative-control (included non-template control) and positive control were performed.

Pear1 Variants Analysis
Ampli cation Refractory Mutation System (ARMS-PCR) Two intronic PEAR1 variants (rs12041331and rs12566888) associated with platelet hyper-reactivity were selected according to literature, and the sequence was retrieved from the NCBI database (16). Because these polymorphisms are due to the single nucleotide changes in the alleles (rs12566888; G/T and rs12041331; G/A), the ARMS-PCR technique was considered a reliable method to detect these point mutations. This technique can also differentiate between heterozygous and homozygous individuals in terms of a single gene locus. For this purpose, two parallel reactions were performed in two separate tubes. In both of these reactions, a similar DNA sample was used. The rst reaction (in tube 1) contained primers speci c for normal DNA (normal forward/reverse primers) that could not replicate the mutated DNA at the gene locus. Conversely, the second reaction (in tube 2) contained a mutant allele-speci c primer and normal primer (normal forward/mutant reverse), so it could not amplify normal DNA. We used three types of primers; Normal forward primer was constant, and its complementary sequence was present in both reactions. This primer was designed for regions of the gene that often lack mutation and serve as internal controls. The normal band was observed in all tubes containing normal primers. The normal tube (containing regular primers) is a kind of biological control in this study to verify ARMS-PCR results' accuracy. The other two types of primers are different at the 3 'end. The rst (normal reverse primer) is speci c to the natural DNA sequence, and the second (mutant reverse primer) is speci c to the mutated nucleotide. ARMS primers were designed by Batch Primer 3 software, which is freely available online at (http://probes.pw.usda.gov/cgi-bin/batchprimer3/batchprimer3.cgi). The mismatch was deliberately added near the 3 'end of the primer (at the terminal and proximal nucleotides) to prevent nonspeci c replication of normal DAN by the mutant primer and subsequently increase the accuracy of the ARMS reaction. In this regard, if a strong mismatch (C-C, G-A, A-A) was observed at 3' end of the primer, a weak mismatch (T-T, T-C, T-G, G-G, A-C) was deliberately added to the nucleotide at -2 position and vice versa. Characteristics of the examined rs12041331/rs12566888 variants and sequences of associated primers are presented in Table 2. The PCR reaction was performed for both variants using the FlexCycler Thermocycler. ARMS PCR reaction for both rs12041331and rs12566888 variants was optimized to amplify the desired region in two separate tubes (for each sample) as follows; 0.5 µl of DNA was used as a template in 10 µl reaction mixture (containing dNTPs, 1× PCR buffer, MgCl2, and Taq polymerase), 0.25 µl of forward and reverse primers and 14 µl of distilled water (the total volume of the reaction solution for each tube was 25 µL). The ARMS PCR procedure for rs12566888 variants consisted of denaturation at 95 C for 5 min, followed by 35 cycles of 95 C for 45 sec, 58 C for 45 sec, 72 C for 1 min, and a nal extension at 72 C for 3 min. Besides, the ARMS PCR procedure for rs12041331variant was performed as follows; initial denaturation at 98 C for 2 min was followed by 35 cycles of denaturation at 98 C for 15 sec, annealing at 61 C for 45 sec, and extension at 72 C for 1 min, the nal extension step was at 72 C for 3 min. After the ARMS-PCR reactions, 6 µl of PCR product was loaded on 2 % agarose gel in ×1 trisborate-EDTA buffer (PAYA PAZHOOHESH, Iran) for electrophoresis, which was stained with DNA safe stain.
Electrophoresis was run in 115 voltage for 50 min. The gel was then inserted into a gel documentation system (under UV light) and analyzed by UV-pro software. Schematic representation of ARMS-PCR assay along with gel electrophoresis of the normal, heterozygous, and homozygous subjects for both rs12041331/rs12566888 variants are shown in Fig. 3.

DNA sequencing for validation of ARMS-PCR ampli cation
The amplicons generated in dual PCR reactions were employed for direct sequencing using the indicated primers and ARMS-PCR conditions to validate ARMS-PCR ampli cation results. For this purpose, some representative genotypes of each variant were sequenced by the Sanger method; However, DNA sequences of only two samples are presented in Fig. 4. All the DNA samples were sequenced based on the following protocol: 0.7 µL Big dye, 3 µL H2O, 4 µL buffer, 5 µl of forward and reverse primers, and 5 µL formamide. Sanger sequencing was performed on an ABI 3130XL Genetic Analyzer (USA), and the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) was used for DNA analysis. After analyzing the sequence data by the UGENE software, the results obtained from DNA sequencing and ARMS-PCR assay were consistent with each other.

Statistical analysis
This study's variables included quantitative and qualitative data, which were reported as mean ± standard deviation (mean ± SD) to describe the central tendency and interquartile range to describe data dispersion. In this research, the t-test and Mann-Whitney test were used to compare the scores of means, and ANOVA was employed to determine the correlation between PEAR1variants (rs12041331and rs12566888), platelet count, WBCs counts, and Hb levels. A chi-squared test was used to compare categorical variables and the determination of the relationship between the case and control data. In all the tests, the p-value was considered less than 0.05 (P-Value < 0.05) as statistically signi cant, and the Odds ratio (OR) with a 95% con dence interval (95%CI) was calculated. All the statistical analyses were processed by SPSS software (statistical package, version 24).

Results
In this study, 105 ET patients with an average age of 60.33 years (± 12.31 SD) and the same number of healthy controls were evaluated for the presence of rs12041331and rs12566888 variants (Table 1). These two variants are localized in intron 1 of the PEAR1 gene (chromosome 1), which, according to previous studies, accounts strongly for platelet function phenotypes and aggregation ( In this study, the mean platelet count in ET patients was 835.33 ×10 9 /L ± 284.56 SD; The mean platelet count in patients with rs12566888 and rs12041331 variant was 920.100 ×10 9 /L ± 272.10 SD and 910.500 ×10 9 /L ± 270.02 SD, respectively. The results reveal that between these two PEAR1 gene variants, rs12041331 is signi cantly associated with increased platelet count in ET patients (P-Value: 0.02, df: 2). is higher compared to CALR + patients (7.29 ×10 9 /L ± 1.45 SD).
Thrombotic events were evaluated as a well-known hematological complication of ET in this study. The prevalence of thrombotic events in ET patients was 29.8%, which was more common in women (64.7%) than men (35.3%). Although thrombotic symptoms were detected in 48.1% of ET patients with rs12041331 variant, no signi cant association was found between thrombotic events and this variant (P-Value: 0.085, df: 2), which was probably due to the small statistical population. Similarly, the prevalence of thrombotic events in patients with rs12566888 variant was 41.9%, that no signi cant relationship was found between the thrombosis and rs12566888 variant (P-Value: 0.176, df: 2). The prevalence of thrombotic attacks in JAK2 + patients was 43.8%, while this frequency in triple-negative and CALR + patients was 16.7% and 15.0%, respectively. In addition to the higher prevalence of thrombotic events in JAK2 + patients compared to triple-negative patients and other ET-related mutations, only a signi cant relationship was observed between this mutation and thrombotic events (P-Value: 0.02, df: 1). Another hematological symptom in ET patients is hemorrhage, which occurs due to platelet dysfunction in the coagulation system despite the high platelet counts. Studies have revealed that in ET patients, hemorrhage is less frequent than thrombosis (3 to 18% of patients) (23) ;In the present study, the incidence of hemorrhage in ET patients was 2.6%, which was more in women (66.7%) than in men (33.3%).

Discussion
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 myelo brosis 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 lifethreatening 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 identi ed 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. Fc RIα (IgE receptor) acts as a ligand for PEAR1, which their connection stimulates platelet aggregation and degranulation (12,35,36). Out of ve 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 identi ed in the PEAR1 gene, such as rs41299597, rs3737224, rs41273215, rs82242, rs11264579, rs12041331, and rs12566888, which increase PEAR1 expression and signi cantly 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 rst 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 rst identi ed ET patients based on a 2016 WHO classi cation through hematological/clinical ndings 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 con rmed by DNA sequencing), and ET-related mutations were identi ed with the Allelespeci c 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 signi cant 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 signi cant 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 con rms 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 signi cantly 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 con rms 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)(46)(47)(48)(49)(50) in the mice models re ected 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, in uences 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.

Conclusions
The results of this study revealed for the rst time that the rs12041331 variant was signi cantly associated with increased platelet count in ET patients. As the prevalence of thrombosis is high in JAK2 + patients, the rs12041331 variant was signi cantly related to thrombotic events. Also, this study, like recent studies, emphasized that platelet counts in JAK2 + patients are lower than in CALR + patients. In contrast, WBC counts, Hb levels, and thrombosis incidence in JAK2 + patients are higher than CALR + patients. Con ict of interest: Dr. Najmaldin Saki (Corresponding author) declares that he has no con ict of interest.
Mohsen Maleknia (First author) declares that he has no con ict of interest. Dr. Mohammad Taha Jalali declares that he has no con ict of interest. Dr. Gholam Abbas Kaydani declares that he has no con ict of interest. Dr. Ahmad Ahmadzadeh declares that he has no con ict of interest. The authors declare no con ict of interest.

Authors' Contributions
Dr. Najmaldin Saki conceived the manuscript and revised it. Mohsen Maleknia and Dr. Mohammad Taha Jalali wrote the manuscript and prepared Tables. Dr. Gholam Abbas Kaydani and Dr. Ahmad Ahmadzadeh were included in qualifying the concept and design. Mohsen Maleknia critically evaluated the intellectual contents. All authors participated in preparing the nal draft of the manuscript, revised the manuscript, and critically assessed the academic contents. All authors have read and approved the manuscript's content and con rmed the accuracy or integrity of any part of the work.    Sequence analysis of CALR exon 9 (52-bp del) and MPL exon 10 (codon W515) in ET patients. Sequence analysis of CALR exon 9 and MPL exon 10 in ET patients. a, 52-base pair deletion is evident in the CALR locus (19p13.13). b, the mutated nucleotide at the codon W515 of MPL locus is visible (1p34.2). Abbreviations: A: Adenine; G: Guanine; C: Cytosine; T: Thymine; del: deletion; bp: base pair.