Molecular markers associated with thrombotic events in Ph-negative myeloproliferative neoplasms

Introduction

Significant progress has been made in understanding the molecular basis of myeloproliferative neoplasms (MPNs) through the identification of several genetic defects, with the most significant being somatic mutations in the JAK2, CALR, and MPL genes. These mutations, which are associated with the molecular pathogenesis of these diseases, affect proteins that play a crucial role in transmitting proliferative signals and are a hallmark of this group of hematopoietic system disorders. JAK2 V617F mutation is found in approximately 90–95% of patients with polycythemia vera (PV), 50–70% of those with essential thrombocythemia (ET), and 40–50% with primary myelofibrosis (PMF) (1-5). Mutations in the CALR gene occur in up to 67–88% of ET and PMF patients with non-mutated JAK2, while mutations in the MPL gene occur in only 5–10% of these patients and are exceedingly rare in PV patients (6,7). It is known that myeloproliferative disorders are often accompanied by adverse vascular events, such as arterial and venous thrombosis and bleeding. These events are most commonly associated with the V617F mutation in the JAK2 gene, rather than mutations in CALR or MPL genes (8-12). On the other hand, mutations in genes that code for blood clotting factors and enzymes involved in the folate cycle and fibrinolytic system, can increase the risk of thrombosis and its recurrence. These include mutations in prothrombin (FII) gene and factor V (FV) gene (13,14).

The Leiden mutation (FV G1691A) has been linked with resistance to protein C anticoagulant effects. The FII G20210A polymorphism, on the other hand, is associated with higher levels of plasma prothrombin. A substitution of guanine for adenine at the 20,210 position in the 3’-untranslated region of the FII gene increases mRNA abundance, ultimately leading to an increase in prothrombin concentration. These two genetic variations are considered to be more common causes of thrombophilia than deficiencies in antithrombin, protein C, or protein S (15-17).

There is another variant of the FII gene mutation—FII Thr165Met (T165M, C494T, rs5896), which is located in a highly conserved region of exon 6. There are limited data regarding the association between T165M mutation and the occurrence of thrombotic complications. However, it was found that the presence of the C494T allele in the FII gene among patients with coronavirus disease 2019 (COVID-19) significantly increased the risk of thrombotic events despite taking anticoagulant medication (18-20).

The high prevalence of genetic markers for hereditary thrombophilia among the general population suggests that a significant number of patients with MPN may have these markers. However, studies on the potential impact of these polymorphisms on thrombotic risk among MPN patients have yielded conflicting results (21,22).

The aim of our study was to evaluate the impact of genetic markers for hereditary thrombophilia on the risk of thrombotic events in patients with MPNs. We present this article in accordance with the STREGA reporting checklist (available at https://aob.amegroups.com/article/view/10.21037/aob-24-35/rc).

Methods

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Ethics Committee of the National Medical Research Center for Hematology, Moscow, Russia (protocol code 180; date of approval 30.05.2024) and individual consent for this retrospective analysis was waived.

Patient selection and data collection

Samples of patients who attended the National Medical Research Center for Hematology between November 2016 and February 2023 were included in a retrospective study. The structure of datasets used for analysis is presented in Figure 1. DNA isolated from the blood samples of 4,197 patients were tested for hereditary thrombophilia markers (FV G1691A, FII G20210A). Electronic data records for these patients were searched for the occurrence of certain “thrombotic” keywords, including: thrombosis, pulmonary embolism, thromboembolism, venous thromboembolic complications, postthrombophlebitic disease, portal hypertension, heart attack, ischemic type of stroke, acute cerebrovascular accident, occlusion, thromboextraction, thrombolytic therapy, thrombolysis, transient ischemic attack, bone necrosis. The search yielded a group of patients [439] whose medical records contained specific keywords. After a thorough analysis of appropriate medical records by clinical experts, 43 patients of these 439 were excluded from the analysis since thrombotic events were not confirmed.

Figure 1 Structure of the dataset. MPN, myeloproliferative neoplasm.

Requests for JAK2, CALR, and MPL evaluation according to the World Health Organization (WHO) 2022 guidelines (23) might be interpreted as a possible indication of MPN presence. Therefore, we have identified 767 patients who were tested for the mutations in the JAK2, CALR, and MPL genes as a group suspected of having MPNs. After an expert evaluation of their clinical records, MPN diagnosis was confirmed for 367 of these patients, and for the remaining 400 patients, MPN diagnosis was excluded. The group with a confirmed MPN diagnosis consisted of 139 men and 228 women. The median age of the patients was 48 years, with a range from 18 to 84 years.

Molecular genetic analysis

DNAs and RNAs were extracted from 5 to 10 mL of blood or bone marrow using standard salt extraction method (24) or “Ribosol-D” kit (Interlabservice, Moscow, Russia). The JAK2 V617F mutation was detected using a reagent kit from Syntol (Moscow, Russia). MPL W515L/K mutations were assessed using a reagent kit also from Syntol. The Rotor-Gene (Qiagene, Hamburg, Germany) apparatus was used for polymerase chain reaction (PCR) amplification. Exon 9 deletions/insertions in the CALR gene were analyzed using fragment analysis with a minimum sensitivity of 3%. All samples were tested for Ph negativity with a combination of reagent kits “Reverta-L” and “Amplification Variant FRT” (Interlabservice). The analysis of genetic mutations in thrombophilia markers, including FII G20210A, FV G1691A, as well as FII Thr165Met, was performed using allele-specific PCR (AS-PCR) on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). AS-PCR conditions: preheating: 94 ℃, 300 s; thermal cycling: 45 cycles; denaturation: 94 ℃, 20 s; annealing and elongation: 60 ℃, 50 s; each primer quantity 10 pm/per reaction; each probe quantity 5 pm/per reaction; reaction volume: 25 µL. PCR master mix (buffer, MgCl₂, dNTP, Taq-polymerase) was provided by Syntol. All primers and Taqman probes were synthesized by Syntol according to our design (see Table 1).

Statistical analysis

All three datasets (genotypes of thrombophilia markers, history of thrombotic events, and the presence of MPN diagnosis) were merged into a single analysis file by patients ID (see table available at https://cdn.amegroups.cn/static/public/aob-24-35-1.xls). The indicator for thrombotic events was set to 0 for all patients except those who had such an event, which were included in a second data set as confirmed by an expert physician. Patients treated at our center receive a comprehensive range of diagnostic and treatment services in accordance with internationally recognized clinical guidelines. Therefore, myocardial infarction was confirmed by electrocardiogram, deep vein thrombosis by ultrasound, pulmonary embolism by tomography, etc. The demographic characteristics of the study sample are presented in Table 2. It should be noted that, despite the initial analysis of the clinical data set was conducted using computer-assisted keyword searching, only cases that were manually verified by clinical experts were included in the subsequent association analysis. Clinical and laboratory records were extracted from the medical and laboratory databases and imported into Statistical Analysis Software (SAS) datasets. These datasets were then sorted, processed, and combined into an analysis file using DATA Step programming tools in SAS (25). Descriptive statistics and frequency analyses were performed on the combined data sets in order to estimate associations.

Results

We have found no essential difference in the occurrence of hereditary thrombophilia gene mutations between our general group of patients (n=4,197) and the data published elsewhere (26-28). The complete genotype distribution for 4,197 patients is provided in Table S1. FII G20210A, FV G1691A, and FII Thr165Met variant alleles were found in 3.14%, 5.17%, and 31.86%, respectively. Of the 4,197 patients, 396 (9.44%) had a history of thrombotic events, including heart attacks, thrombosis, PE, and other circulatory disorders. The distribution of 367 patients with a confirmed diagnosis of MPNs established according to the WHO diagnostic criteria (29,30) is as follows: PV 27.79% (n=102), ET 17.98% (n=66), PMF 16.35% (n=60); MPN not otherwise specified 37.87% (n=139). Thrombotic events in patients with the JAK2 V617F gene mutation were observed in 17.3% (44/255). Thrombotic events in patients with CALR and MPL gene mutations occurred in 7.5% (4/53) and 50% (3/6), respectively. No significant differences were found in the occurrence of FII G20210A, and FV G1691A gene mutations between the general population and patients with confirmed MPNs. Treatment for MPNs involves assessing thrombotic risk to prevent thrombotic complications. Therefore, it is plausible that prescribed anticoagulant therapy compensates for the prothrombotic effects of hereditary thrombophilia gene mutations in these patients. Additionally, the data were analyzed for the group of patients suspected of having an MPN (n=767). We found a synergistic effect between MPN diagnosis and the presence of the FII C494T (Thr165Met) mutation. The risk of thrombotic complications in these cases was nearly twofold higher compared to patients without an MPN diagnosis (Figure 2). This difference was statistically significant [21.6% vs. 12.4%; P_χ2=0.02; odds ratio (OR) =1.95; 95% confidence interval (CI): 1.10–3.45]. In the overall cohort of patients (n=4,197), no significant correlation between the FII Thr165Met mutation and thrombotic events was found (P_χ2=0.70; OR =1.05; 95% CI: 0.83–1.34) (Tables 3,4).

Figure 2 Association of thrombotic events with thrombophilia molecular markers (FII T165M, FII G20210A, and FV G1691A) in patients with MPNs. CI, confidence interval; FII, prothrombin; FV, factor V; MPN, myeloproliferative neoplasm; OR, odds ratio.

Discussion

Currently, limited data are available regarding the clinical significance of the FII C494T mutation (Thr165Met). The impact of this mutation on the development of urolithiasis, Usher syndrome, and miscarriage has been confirmed in several studies (31-34). A relationship between this polymorphism and familial thrombosis has also been reported (19,20). These studies describe patients who are homozygous or heterozygous for the FII Thr165Met mutation, leading us to evaluate both heterozygous and homozygous variants of this mutation as potential genetic markers for thrombophilia.

The prothrombinase complex, which includes phospholipids, Ca²⁺ ions, and coagulation FV and factor Xa plays a key role in coagulation by converting prothrombin into thrombin. Coagulation factor Xa is a proteolytic enzyme that acts as the active component of prothrombinase. This enzyme cleaves approximately half of the prothrombin molecule, causing the loss of 3/4 of its carbohydrates. Thrombin has both pro- and anti-coagulant properties, making it an important regulator of blood clotting. Exon 6 of the FII codes for a Kringle domain, which plays a crucial role in protein-protein interactions involved in blood clotting. The C494T mutation in FII exon 6 results in the substitution of the amino acid threonine (Thr) with methionine (Met) at position 165 (35,36). This amino acid substitution can affect interactions between proteins involved either in coagulation or anticoagulation. Antithrombin III is one of these factors. It can be speculated that changes in the functional domain, specifically those caused by the C494T (Thr165Met) mutation, prevent the thrombin-antithrombin III interaction and can lead to thrombosis, even in the presence of anticoagulants.

Here we present data concerning a synergistic effect between MPN diagnosis and the Thr165Met mutation in the FII gene on thrombosis. However, our study has certain limitations. The main limitation was that only past medical events were tracked and confirmed based on medical records. We understand this, but we used all available information derived from existing medical records for this retrospective research. It should also be noted that the control group, which was formed from patients at the same hematology center, does not represent a general population. However, this approach was deemed appropriate for the study’s objective, which was to compare two patient groups from the same clinical setting, differing primarily by MPN status. Selection bias was minimized, as we included all patients who had thrombophilia markers tested during the specified time period. Group formation was also based on objective parameters and was not influenced by the researchers.

Therefore, a possible link is assumed between the presence of the C494T mutation (Thr165Met) in the FII gene and thrombosis in patients with MPNs who are receiving anticoagulant therapy. Further research should clarify this association and help determine optimal anticoagulant treatment strategies in these patients.

Conclusions

The presence of the Thr165Met mutation in the FII gene was found to be associated with an increased risk (OR =1.95; P_χ2=0.02) of thrombotic events in patients MPNs. It should be noted that the high incidence of Thr165Met variant allele (31.86%) indicates that our findings need to be confirmed on a larger sample of patients. However, the significance of certain molecular markers as predictive factors for assessing thrombotic risk and prescribing appropriate antiplatelet and anticoagulant therapy in patients with Ph-negative MPNs should not be overlooked.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STREGA reporting checklist. Available at https://aob.amegroups.com/article/view/10.21037/aob-24-35/rc

Data Sharing Statement: Available at https://aob.amegroups.com/article/view/10.21037/aob-24-35/dss

Peer Review File: Available at https://aob.amegroups.com/article/view/10.21037/aob-24-35/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://aob.amegroups.com/article/view/10.21037/aob-24-35/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Ethics Committee of the National Medical Research Center for Hematology, Moscow, Russia (protocol code 180; date of approval 30.05.2024) and individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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