Molecular landscape of the JAK2 gene in chronic myeloproliferative neoplasm patients from the state of Amazonas, Brazil
- Authors:
- Published online on: October 23, 2023 https://doi.org/10.3892/br.2023.1680
- Article Number: 98
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Copyright: © Torres et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
The BCR::ABL1 negative chronic myeloproliferative neoplasms (MPN) represent a heterogeneous group of clonal diseases of the hematopoietic progenitor cell, of which the most classic are polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) (1,2). In the 5th Classification of Hematolymphoid Tumors, published in 2022, the World Health Organization (WHO) revised certain aspects for the category of MPN (1), establishing as diagnostic criteria for the diagnosis of PV elevated hemoglobin concentration and/or hematocrit, accompanied by panmyelosis and detection of JAK2V617F or exon 12 variants in JAK2.
The primary diagnostic criterion of ET is marked thrombocytosis (platelet count >450x103/mm3). PMF is characterized by a proliferation of abnormal megakaryocytes and granulocytes in the bone marrow, which is associated in fibrotic stages with a polyclonal increase in fibroblasts that drive secondary reticulin and/or collagen marrow fibrosis, osteosclerosis, and extramedullary hematopoiesis. Thereby, these diseases are characterized by increased cell proliferation, development of chronic inflammation, and association with clonal hematopoiesis (1,3,4).
Missense mutations in the JAK/STAT pathway are the primary causes of the development of chronic MPN (5). Variants in the driver genes JAK2, CALR, and MPL are the most commonly associated with the development of MPN (6). According to National Center of Biotechnology Information (NCBI:https://www.ncbi.nlm.nih.gov/gene/3717), the JAK2 gene is located on chromosome 9p24.1 and encompasses 145,559 nucleotides, distributed across 28 exons, and the JAK2 coding sequence has a length of 3,399 nucleotides, distributed across 23 exons, from exon 3 to exon 25, which encodes a protein of 1,132 α. amino acids, a non-receptor tyrosine kinase named JAK2.
Most of the variants identified in JAK2 result in a gain of function, and are characterized as somatic missense types that lead to unregulated production of hematopoietic cells in bone marrow and accumulation of mature cells in peripheral blood (7). JAK2V617F (dbSNP: rs77375493) is the most commonly identified variant in MPN and is found in up to 95% of cases of PV and between 50-60% of cases of ET and PMF (8). This variant is located in exon 14 of the JAK2 gene and is characterized as a missense variant. It is a product of the substitution of a guanine by a thymine at position 1,849, that leads to a substitution of valine with phenylalanine at the amino acid position 617 (V617F) of the protein structure (9,10), a position that belongs to the pseudokinase domain, which is a region of the primary positive and negative regulation of the protein (10,11).
Variants in exon 12 of the JAK2 gene are identified in ~3% of JAK2V617F-negative patients diagnosed with PV (12). Genetic alterations in this exon include missense and indel variations (13), which confer a marked erythrocytic picture in individuals with PV, and appear at younger ages when compared to the JAK2V617F variant (14).
The presence of coexisting non-driver variants can modulate the JAK2V617F variant allele frequency (VAF). In MPN, the determination of JAK2V617F VAF is pivotal when evaluating laboratory and clinical implications. It is worth mentioning that, in PV, a high VAF (≥50%) is associated with fibrotic progression and positively associated with total white blood cell count (WBC), neutrophil count, and thrombosis events, especially in the presence of coexisting non-driver variants (15), while in ET, a high VAF is correlated with increased thrombo-hemorrhagic events, hypercoagulable status, and low quantitation of hemostasis factors (16,17).
Sanger sequencing and next-generation sequencing have allowed the identification of variants in other JAK2 exons (18,19). Several variants have been identified in the complete coding region of the JAK2 gene, which affect other domains of the JAK2 protein (19,20) and lead to constitutive activation of the JAK/STAT pathway, with most of the described variants being somatic, with only a small fraction of them being germinal. This finding suggests that certain patients may develop a non-clonal myeloproliferative phenotype, with variable penetrance at the familial level (21).
Certain variants that are acquired in the coding region of JAK2 are described as benign or of uncertain clinical significance, and the primarily affected exons are 6(22), 9-10(23), 11-15(19), and 19(24). According to certain studies, some variants in these regions have been found in coexistence, presenting cytokine-independent signaling (25), and are even associated with leukemic transformation and development of non-hematological solid tumors (23,24,26). Thus, the present study aimed to molecularly characterize variants in the complete coding region of the JAK2 gene in individuals with BCR::ABL1 negative chronic myeloproliferative neoplasms.
Materials and methods
Patients
In the present study, 97 patients from the state of Amazonas, Brazil, diagnosed with PV (n=38), ET (n=55) and MF (n=04), who were treated between July 2021 and March 2023 at Hospital Foundation for Hematology and Hemotherapy of Amazonas (which is the only reference institution in the state of Amazonas for the diagnosis and treatment of hematological diseases) were included. Participants showed an absence of BCR::ABL1 transcripts. Additionally, all the patients with a MF diagnosis who agreed to participate in the investigation were included.
The prese study was performed in accordance with the Declaration of Helsinki and Resolution 466/12 of the Brazilian Ministry of Health. This study was approved by the National Ethics Committee, which is responsible for approving relevant human studies in Brazil (approval no. 4.450.813). Written informed consent was obtained from all subjects involved in the study.
Clinical and laboratory data
Clinical data were obtained from medical records, which included data regarding sex, age, splenomegaly, history of thrombotic or hemorrhagic events, and treatments administered. Laboratory data were obtained from blood samples and included red blood cell count (RBC), hematocrit (Ht), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), WBC, percentage of segmented neutrophils, monocytes and lymphocytes; Platelet count, prothrombin time-International Normalized Ratio (PT-INR), activated partial thromboplastin time (aPTT), fibrinogen (FIB), lactate dehydrogenase (LDH) and uric acid (UA). UA and LDH analyses were performed after diagnosis and during treatment, mentioning that several patients included in the study had received several years of hydroxyurea administration. The median optimal treatment regime in PV patients was 4 years (100-500 mg/per day of hydroxyurea or 2 mg/per day of Anagrelide), in ET patients it was 10.5 years (100-300 mg/per day of hydroxyurea or 2 mg/per day of Anagrelide), and in MF patients it was 2 years (2 mg/per day of Anagrelide). Of note, administration of hydroxyurea can significantly alter laboratory analysis.
Blood-sample processing and RNA extraction
Total RNA was extracted from peripheral blood samples with EDTA anticoagulant using TRIzol® (Ambion; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. cDNA was synthesized using SuperScript™ III Reverse Transcriptase (Promega Corporation). Reverse transcription was used to obtain cDNA, using the following thermocycling parameters: 5 min at 25˚C and 60 min at 42˚C. After the reaction, the cDNA was stored at -80˚C until used for PCR.
PCR and Sanger sequencing analysis
Amplifications were performed using a total volume of 25 µl. Reaction products were visualized using electrophoresis on a 1.5% agarose gel stained with ethidium bromide. PCR products were purified with the DNA precipitate and purification protocol using polyethylene glycol 8000 (Promega Corporation) as described previously (27-29). A Sanger sequencing reaction (in both directions) was performed using BigDye® Terminator v3.1 (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The sequences of the primers used are listed in Table I, and were designed using Primer-BLAST-NCBI and OligoAnalyzer Tool-IDTDNA to evaluate the percentage of GC, Tm, Hairpin capacity, and ΔG index, to flank the complete coding region of JAK2, spanning from exon 3 to exon 25 (Fig. 1). The products of the sequencing reaction were purified using the EDTA/ethanol protocol and were subsequently evaluated in an automatic sequencer (3500 XL Genetic Analyzer®, Applied Biosystems handbook; Thermo Fisher Scientific, Inc., pag. 12) using the POP-7 polymer.
Data analysis
The sequences obtained were initially analyzed using the Sequencing Analysis software (Applied Biosystems; Thermo Fisher Scientific, Inc.); only high-quality sequences were used for variant analysis (Q score ≥30). Geneious software 6.0.6 (Biomatters, Inc.) was used to obtain contigs and compare them to the Homosapien JAK2 reference sequence, transcript 2, mRNA (NCBI: NM_001322194.2). Samples with the presence of rare variants were sequenced and confirmed at least twice. VAF was measured in JAK2V617F-positive individuals using Minor Variant Finder (Applied Biosystems, Thermo Fisher Scientific, Inc.) and Edit R software (moriaritylab.shinyapps.io/editr_v10). The clinical significance of the variants identified in the research was analyzed using the Polyphen2 tool and the ClinVar-NCBI site (https://www.ncbi.nlm.nih.gov/clinvar/).
Statistical analysis
Categorical variables are presented as the frequency (n, %). Continuous numeric variables are presented as the median and interquartile range (IQR). The distribution of continuous numerical variables was verified using a Shapiro-Wilk test. Statistical analysis of categorical variables was performed using a00202 test. Kruskal-Wallis and Mann-Whitney U tests were used to analyze numerical variables, when appropriate. Data from individuals with MF were excluded from the statistical analysis between groups due to the number of patients with MF. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis of the data was performed using GraphPad Prism version 8.2.1 (GraphPad Software, Inc.).
Results
Clinical and laboratory characteristics of patients
Samples from 97 patients diagnosed with MPN were evaluated, and these were distributed among PV (n=38), ET (n=55), and MF (n=04). During the length of the study, none of the patients showed transformation to acute leukemia, post-PV, or post-ET-MF. Clinically, ET showed a predominance in females (P=0.0276), compared with PV and MF. All individuals were between the fifth and sixth decade of life (P=0.565; comparing the age between the PV and TE groups). Splenomegaly was detected more frequently in MF, than in PV and ET (75, 23.6, and 16.3%, P=0.0212, respectively) patients.
Thrombotic and hemorrhagic events were more often observed in ET cases (16.3 and 21.8%, P=0.6406 and P=0.0205, respectively) when compared to PV cases. The thrombotic events included deep venous thrombosis, thrombosis of the splenic vein, esophageal varices, and miscarriage, and the following hemorrhagic events were evaluated in the study: Hypermenorrhagia, ocular and gingival hemorrhage, and hemorrhage of the gastrointestinal tract. All medical records of the patients included in this study were reviewed and none of these reported acquired von Willebrand syndrome.
In the blood count, an increase in the erythrocyte lineage was observed in individuals with PV compared to those with ET and MF, with an increased RBC (5.03 x mm3, P<0.0001), a finding that is complemented by Ht values (48%, P<0.0001) and Hb concentration (15.2 g/dl, P<0.0001). Hemometric values were found to be increased in ET cases [Mean Corpuscular Volume, MCV: 103.9 fl; P=0.0013; Mean Corpuscular Hemoglobin, MCH: 33.5 pg, P=0.006, and Mean Corpuscular Hemoglobin Concentration (MCHC): 32.5 g/dl, P=0.1160] when compared to PV and MF cases. The white blood cell count was within normal ranges in PV and TE cases, compared with those with MF (P=0.0134). However, the percentage of neutrophils was higher in MF patients (76.4%) when compared to ET and PV patients (P=0.0232), and the lymphocyte count was slightly higher in ET than in PV and MF patients (29.2%, P=0.0005). In ET patients, a high platelet count was observed when compared to PV and MF patients (470,500 x mm3, P<0.0001). Erythropoietin measurements were not available in the present study.
Values in the hemostasis tests of individuals with PV, ET, and MF were closely related; however, a slight increase in fibrinogen concentrations was observed in individuals with MF (321 mg/dl, P=0.400). Biochemical analyses demonstrated higher concentrations of LDH and UA in subjects with MF (904.5 U/l, P=0.0295 and 6.8 mg/dl, P=0.006; respectively) compared with PV and ET patients. Clinical and laboratory values are described in Table II.
Variants detected in chronic MPN patients
In this study, missense variants were identified in the FERM domain (rs907414891); 1 variant in the FERM-SH2 linker region (rs2230723), 1 variant in the pseudokinase domain (rs77375493), and 1 variant in the kinase domain (rs41316003). This totals 4 missense variants identified in the complete coding region of the JAK2 gene, as described in Table III. In addition, other synonyms and benign variants were detected in the complete coding region of the JAK2 gene (rs2230722, rs576746768, rs2230728, rs2230724, and rs55930140). Conversely, the rs10119726 variant is a synonymous variant and does not have a description of its clinical significance on ClinVar. These variants are shown in Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10 and Fig. 11.
 | Table IIIMissenses variants detected by Sanger sequencing in the entire coding region of the JAK2 gene in patients with myeloproliferative neoplasms. |
Frequency and distribution of missense variants in patients with variant alleles of the JAK2 gene
The frequency of variants was estimated in the population (PV=38, ET=55, and MF=04), and it was noted that most of them were in the first protein domains, especially in the FERM domain, followed by the pseudokinase domain. The variant rs77375493 (JAK2V617F) showed a high frequency in individuals with PV when compared to those with ET (65.7 and 38.1%, respectively, P=0.0116). Variant rs2230723 was found in sporadic cases of PV and ET. Interestingly, rs907414891 and rs41316003 were found only in cases of ET, but not in cases of PV or MF. The frequency of missense variants is presented in Table IV.
Mutational landscape of the JAK2 gene in individuals with chronic MPN
After estimating the frequency of the variants in the complete coding region of the JAK2 gene, the mutational profile of the individuals was mapped. It was observed that patients with variant alleles in JAK2 simultaneously presented with 1-3 variants. Among the primary variants found simultaneously in the three types of MPN were rs2230724, rs2230722, and rs77375493, thus highlighting that most individuals with PV presented with the three variants when compared to those with ET (P=0.0023). In contrast, individuals with ET showed a predominance of two variants (rs2230722 and rs2230724) compared to those with PV (P=0.0253). The mutational landscape of the patients is presented in Table V. Individuals with four variants were not found.
JAK2V617F VAF in patients with PV and ET
Of the 97 patients included in this study, the allele burden of JAK2V617F was measured in 46 individuals who were JAK2V617F-positive (PV, n=25 and ET, n=21). The allele burden of JAK2V617F was compared in individuals with PV and ET. In each disease, two groups were considered to describe the VAF of JAK2V617F: High VAF (≥50%) and low VAF (<50%). Individuals with ET showed a low VAF JAK2V617F (<0.0001) when compared to those with PV who showed VAF ≥50% (0.0477). Individuals with MF were excluded from this comparison. The comparison of the VAF of JAK2V617F among the groups is presented in Table VI.
Comparison of the clinical and laboratory profile according to the VAF of JAK2V617F in patients with PV
The clinical and laboratory profile of individuals with PV and ET with JAK2 variants were compared considering the VAF of JAK2V617F in both groups [high VAF (≥50%) and low VAF (<50%)]. Regarding the clinical profile in individuals with PV, thrombotic and hemorrhagic events were evenly distributed among both groups. However, in the PV patients, splenomegaly was more frequent in individuals with a high VAF. The clinical data of the individuals with PV according to VAF of JAK2V617F are presented in Table VII.
The comparison of laboratory profiles in individuals with PV, according to their VAF of JAK2V617F, showed an increase in hematimetric values (RBC, 4.7 x mm3; Ht, 46.9%; Hb, 14.9) in individuals who presented a VAF of JAK2V617F ≥50%, compared with those with a VAF of <50%. WBC and platelet count were slightly augmented in individuals with a VAF of ≥50%. Likewise, LDH was elevated in individuals with a VAF of JAK2V617F of ≥50% (486.5 U/l). Hemostasis tests were relatively equivalent between both groups in PV patients. The laboratory profiles of the individuals with PV, according to the VAF of JAK2V617F, are presented in Table VII.
Comparison of the clinical and laboratory profiles according to the VAF of JAK2V617F in patients with ET
In the individuals with ET, the clinical and laboratory profiles were also described based on the VAF of JAK2V617F. Regarding the clinical characteristics in individuals with ET, thrombo-hemorrhagic episodes were the most commonly recorded clinical events in the patients, especially in those with VAF of JAK2V617F of ≥50%; however, despite this fact, it was not statically significant. Just as in the PV individuals, splenomegaly was more frequent in individuals with a high VAF. The clinical data of the individuals with ET according to the VAF of JAK2V617F are presented in Table VII.
The laboratory profiles of individuals with ET, according to the VAF of JAK2V617F, showed an increase in hematimetric values (RBC, 5.1 x mm3; Ht, 47.0%; and Hb, 15.5 g/dl) in individuals who presented a VAF of JAK2V617F of ≥50% when compared to those with a VAF of <50%. The WBC showed equivalence in both groups. Interestingly, the platelet count was increased in individuals with a VAF of <50%. Likewise, for individuals with ET, LDH was elevated in individuals with a VAF of JAK2V617F of ≥50% (412.1 U/l). Hemostasis was slightly prolonged in individuals with a VAF of JAK2V617F of ≥50%. The laboratory profiles of individuals with ET, according to VAF JAK2V617F, are presented in Table VII.
Discussion
MPNs are generally characterized by an increase in cell counts in the blood, which can lead to clonal evolution and disease progression. Despite investigations in other Brazilian states (30-32), this study is the first to address JAK2V617F mutation detection and the hematologic profile according to JAK2V617F VAF in patients from the state of Amazonas diagnosed with MPN.
Regarding the proportion of MF patients, which is a multifactorial issue, previous studies in Brazil have shown a lower proportion of MF patients compared with PV and ET (30-32), and it is noteworthy that MF is the most aggressive MPN, and shows a high ratio of leukemic transformation. Silva et al (32) determined the prevalence of JAK2V617F in MPN in Pernambuco, Brazil, and found that few patients had MF diagnosis compared with those with PV and ET. Similarly, Macedo et al (30) investigated the association between the JAK2 46/1 haplotype and acquisition of JAK2V617F. They observed the lowest number of cases of MF. Furthermore, they concluded that the JAK2 46/1 haplotype was present in JAK2V617F positive individuals and associated with MPN phenotype in Brazilian patients. Likewise, in another study, Macedo et al (31) assessed the association of TNF polymorphisms with JAK2V617F MPN in Brazilian patients finding a low number cases of MF.
The present study showed that the increase in the erythrocyte lineage was in fact a characteristic of individuals with PV and that the increase in the platelet count was an indicator that is suggestive of ET, according to the indicators established by the WHO (1). RBC counts are directly related to Hb and Ht concentrations; it is hypothesized that these two hematological parameters are reliable indices for the diagnosis of PV (33).
Currently, erythropoietin measurement is considered a major diagnostic criterion for PV diagnosis (1,34). In the present study, these measurements were not available; however, MCV is considered a marker that can be used to differentiate between PV and ET (33). In the present study, MCV was found to be lower in patients with PV than in those with ET. This finding may explain the iron deficiency and the accelerated time for renewal of red blood cells in these patients (33,35).
The role of the lymphocyte count in MPN is not well described. Stefaniuk et al (36) found that there was little evidence for the prognostic significance of the neutrophil-lymphocyte ratio and lymphocyte-monocyte ratio in MPN, but they both may be higher in patients with PMF compared to healthy individuals, and may be associated with chronic inflammation and tumorigenesis. Likewise, Mulas et al (37) described that high a neutrophil-lymphocyte ratio had been reported in JAK2-positive patients and this parameter could be used as an indicator of chronic inflammation in MPN.
In addition, Vannucchi et al (38) reported that individuals with MPN have an increased risk of developing lymphoproliferative neoplasms, particularly in those that were JAK2V617F-positive. Similarly, Garcia-Gisbert et al (39) found that certain patients with a diagnosis of MPN showed CD3+ JAK2V617F-positive lymphocytes, These findings may support the hypothesis that JAK2V617F-positive lymphocytes may be related to leukemic transformation.
Furthermore, it has been highlighted that MPN is associated with a high risk of thrombotic and thromboembolic events when compared with the general population, and is also associated with increased hematopoietic counts (40), which was also observed in the present study. This fact may be explained by the presence of a high VAF of JAK2V617F (≥50%), which likely stimulates deregulation signaling in hematopoietic progenitor cells and may be potentialized by the presence of other variants in genes such as CALR and MPL; these are directly implicated in platelet activation and increased platelet account (40).
Administration of hydroxyurea is frequently used in cases of PV and ET for the normalization of hematological counts (41,42). The results of the present study showed that the high platelet count observed in individuals with ET was directly related to the increase in the frequency of thrombo-hemorrhagic events, which indicates that platelets could in fact be the primary mediators of thrombotic activation in these patients. As such, the study by Buxhofer-Ausch et al (43) demonstrated that platelet count normalization is an important factor in reducing thrombotic risk, regardless of the leukocyte count. However, further studies are needed to confirm what the cut-off point in the platelet count is to trigger these risks.
Esophageal and gastric complications are often described in patients with myeloproliferative neoplasms diagnosis (44), and this is typically due to portal system hypertension or von Willebrand syndrome, which is the result of excessive thrombocytosis. However, in the present study, bleeding complications were relatively high, especially in patients with ET. This fact may be due to an increased platelet count with functional platelet disorders, such as impaired platelet aggregation response to collagen and reduced number of dense granules in platelets (45). In addition, current literature notes that ET is more common in females, and bleeding and thrombotic risks are the major complications in MPN patients (40,46). Nevertheless, female biology may play a role in the development of bleeding and thrombotic events, likely due to pregnancy and the use of contraceptives interfering with the interactions of platelets and other molecules in the endothelium.
Other variants in the JAK2 gene have been reported, and most of these variants are of the somatic type (21,22). The existence of germline variants in MPN has also been described, and this includes showing patterns of erythropoietin (EPO) hypersensitivity and weak constitutive signaling of the JAK2/STAT5 pathway compared to JAK2V617F (47).
Therefore, by applying Sanger sequencing in the complete coding region of the JAK2 gene, the results of the present study demonstrated the existence of somatic and germline variants in individuals with MPN other than JAK2V617F, with somatic variants being the most frequent. This is also corroborated by previous studies (19,48,49). Moreover, germline variants in individuals with MPN at an early age in individuals with a familial predisposition, compared with those with somatic variants, have been described (50). Age differences between patients with somatic and germline mutations were not investigated in the present study, and this will form a future research direction.
JAK2V617F is the most common variant in BCR::ABL1 negative MPN (51), with constitutive activity of the JAK2/STAT5/STAT3 pathway, and it is highly associated with the development of cardiovascular and thrombotic complications (15). In the present study, JAK2V617F was identified in 65.7% of the patients with a diagnosis of PV. This may be related to the median optimal treatment regimes, as these individuals have been treated with cytoreductive therapy for several years.
The effects of JAK2 VAF are well established; however, the specific populations affected are poorly understood. Through the comparison of JAK2V617F VAF, it was shown that patients from the state of Amazonas with PV had a JAK2V617F VAF that was higher than those diagnosed with ET, and individuals with a VAF of ≥50% had more thrombo-hemorrhagic events and a slight prolongation in coagulation tests, especially in PT-INR and aPTT when compared with those with a VAF of <50%, which is that not dissimilar to previous studies (40,46). This fact directly suggests that individuals with a high JAK2V617F VAF exhibit increased intracellular signaling, cellular activation, and possible alterations in coagulation factors, thus contributing to the deregulation of hemostasis.
Furthermore, the results of the present study are in agreement with the results of Hu et al (16) who demonstrated that individuals with PV had a high JAK2V617F VAF (≥50%) compared with those with ET. In addition, the results of the present study demonstrated that patients from the state of Amazonas with a diagnosis of PV had a mutational landscape that was more complex than that of individuals with ET from the same state. This landscape showed at least three mutations in concomitance in the JAK2 gene, suggesting genomic instability and, subsequently, the instability of regulatory mechanisms at the protein level and possibly in the myeloproliferative phenotype of individuals with MPN.
According to data available on the ClinVar-NCBI website, a number of the acquired variants located in the extension of the JAK2 coding region are either benign or of uncertain clinical significance. This indicates that most of the variants reported to date are in the FERM domains, kinase, and binding regions (19,22-24), and this finding relates to the present study, since the detected variants are located in the aforementioned regions.
Thus, it is highlighted that the presence of variants in the FERM domain may result in increased basal activity of JAK2 (52,53), which is a phenomenon that may explain the myeloproliferative phenotype in JAK2V617F-negative individuals who present with other variants in the JAK2 gene, and could possibly be related to the clinical phenotype in the different subtypes of neoplasms, a phenomenon that is still not well understood. The present study identified the rs907414891 variant, located in the FERM domain, which results in the exchange of isoleucine for valine at position 166 of the JAK2 protein (p. Ile166Val). Currently, this variant has no description in the literature regarding its clinical impact. However, the exclusive presence of rs907414891, rs576746768, rs413160003, and rs55930140 in ET individuals may represent novel clonal biomarkers in ET. Nevertheless, it is necessary to perform additional molecular and functional tests to verify their possible association with MPN.
The SNV rs2230722, located in exon 6 of JAK2, was frequently observed in the present study and had a higher predominance in females, in agreement with Sokol et al (22). This variant was more frequent in women with platelet aggregation syndrome compared to men; and was significantly associated with deep vein thrombosis. As such, the variant could be correlated with the clinical picture of MPN, especially in individuals with thrombotic complications. The SNV rs2230724, a variant that is present in exon 19 of JAK2, was detected in the present study in the JH2-JH1 linker region. Although variants in this region are not frequently described in MPN, alterations in the JH1-JH2 interaction may generate dysregulation in the inhibition of catalytic activity and, therefore, alter its function. This SNV, together with rs2230728, are reported in hematologic cancers and associated with the progression to acute leukemia, especially in individuals older than 45 years old (23); and may thus serve as genetic markers of leukemic progression in MPN.
The coexistence of JAK2 variants is not often described in MPN; however, this could have greater repercussions in the individual's clinical picture (50). In the present study, concomitance was observed in up to three variants, in the presence of JAK2V617F, and presented laboratory profiles with slight increases in cell counts, including red blood cells and platelet counts, which indicates that these variants may confer genomic instability and increase intracellular signaling of the JAK/STAT, PI3K, MAPK, NF-κB, and HIF1-α pathways, to induce tumorigenesis and facilitate the acquisition of other variants within the same gene (50,54).
Using Sanger sequencing, Lanikova et al (55) demonstrated the presence of SNV rs2230723 in coexistence with JAK2V617F and, in this case, described normalized hematological counts after administration of hydroxyurea. In other experiments, both variants showed increased STAT1, STAT3, and STAT5 signaling, which suggested the potential of both variants in the predisposition to malignancies. Likewise, other variants in JAK2 may confer weak constitutive signaling of the JAK/STAT pathway, resulting in a ‘more attenuated’ myeloproliferative phenotype, with slightly altered cell counts. However, further studies are needed to assess the functional behaviors of these variants, both individually and when combined.
Although the present study highlights the importance of detecting other variants in the entire coding region and the coexistence of variants in the same gene with possible repercussions on the clinical and laboratory status of individuals with MPN, it has several limitations. Among the primary limitations of this study is the small sample size due to the lack of patients from various centers. Future studies will aim to recruit a larger cohort from several centers to confirm the results. Here, only patients from the Hospital Foundation of Hematology and Hemotherapy of Amazon were included (a unique reference institution in the state of Amazonas for the diagnosis and treatment of hematological diseases). Another limitation is the lack of functional studies that confirm the myeloproliferative activity of these variants, the lack of allelic association of variants with outcomes, which may explain the possible predispositions for the development of MPN, and JAK2 analysis was performed once along of the study. Likewise, the individuals included in the present study were treated with hydroxyurea and anagrelide, decreasing the probability of the detection of JAK2V617F mutations. The results also may be affected by the low sensitivity of Sanger sequencing.
In conclusion, individuals with negative BCR::ABL1 MPN may present with more than one variant in the JAK2 gene, in particular rs2230722, rs2230724, and rs77375493 variants, both separately and together, and those with a high JAK2V617F VAF show alterations in the clinical-laboratory profiles compared with those with a low JAK2V617F VAF.
Acknowledgements
The authors would like to thank Dr Nadja Garcia Romero (Genomics Laboratory-HEMOAM), Dr Luciana Cassa (Genomics Laboratory-HEMOAM), Rechfy Kasen Abou Ali (MSc.; Genomics Laboratory-HEMOAM) and Dr Enedina Nogueira (Genomics Laboratory-CAM/UFAM).
Funding
Funding: The present study was supported by the Fundação de Amparo à Pesquisa do Estado do Amazonas (Pro-Estado Program; grant nos. #002/2008, #007/2018 and #005/2019, and POSGRAD Program grant nos. #008/2021), Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior.
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request. The GenBank accession nos. for the nucleotide sequences are ON706985 and ON706994.
Authors' contributions
AMT designed the study. DGT, GAVS, LPDSM and AMT prepared the manuscript and performed the literature search. LPDSM, AM, EVBA, MADS, WHL, JP, EA, DC, NAF, RA and LN acquired all the data. AGC, GAVS, AM, EVBA, MADS, WHL, JP, EA, DC, and AMT interpreted the data. AMT, AGC, GAVS, and DGT analyzed the data. AMT, AGC, NAF, RA, LN, and GAVS edited the manuscript. AMT, DGT, GAVS, LPDSM confirm the authenticity of all the data. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
The present study was performed in accordance with the Declaration of Helsinki and Resolution 466/12 of the Brazilian Ministry of Health. The present study was approved by the National Ethics Committee, which is responsible for approving relevant human studies in Brazil (approval no. 4.450.813). Written informed consent was obtained from all subjects involved in the study.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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