Abstract
Traditionally considered to be absent in India, prothrombin gene G20210A (NM_000506.5(F2): c.*97G > A) mutation (PGM) has recently been reported in few Indian patients. We aimed to assess the prevalence of PGM in patients with thromboembolic events from north India region. The thrombophilia workup comprising Protein C, Protein S, Antithrombin functional activity, lupus anticoagulant and anti-ACA and anti-ß2GP1 antibodies were performed in coagulation analyzer (ACLTOP-500, Instrumentation Laboratory, USA) and automated chemiluminescent assay analyzer (ACUSTAR, IL) respectively. PCR–RFLP was used to perform PGM and FVL mutation. Out of 509 patients, DVT and CVT/CSVT were identified in 208 and 250 patients respectively. A total of 42 (8.2%) cases showed inherited thrombophilia and 11 (2.1%) acquired thrombophilia. Among the inherited defects, the most common was FVL mutation 31 (6%) The PGM was seen in only 2/509 (0.3%) patients. The prevalence of PGM in North Indian patients with DVT, stroke and CVT is 0.41% (2/509). Although PGM is rare in this population, its presence emphasizes its association with these conditions. However, the role of PGM testing remains debatable due to its scarcity among North Indians.
Supplementary Information
The online version contains supplementary material available at 10.1007/s12288-024-01741-x.
Keywords: Cerebral venous thrombosis, Deep vein thrombosis, Factor V Leiden, Prothrombin gene (G20210A) mutation, Stroke, Thrombophilia
Introduction
Thrombophilia is a clinical condition characterised by increased risk of blood clots in the veins and arteries. This can lead to a spectrum of clinical manifestations including deep vein thrombosis (DVT), pulmonary embolism (PE) and strokes. Thrombophilia can be caused by genetic factors, acquired conditions, or a combination of both [1]. Inherited disorders or genetic causes of thrombophilia include factor V Leiden (FVL) mutation, prothrombin gene (G20210A) mutation (PGM), deficiency of antithrombin III (AT), protein C (PC) and protein S (PS). Factor V Leiden mutation is the most common inherited form of thrombophilia. PGM is the second most common genetic form of thrombophilia in western world, occurring in about 1.7–3% of the European and US general populations [2–4]. The single base substitution (G-A) at position 20210 leads to polymorphism which is associated with elevated level of plasma prothrombin, a threefold increased risk of venous thromboembolism and a possible increased risk of ischemic strokes [4].
Despite being a well-known genetic risk factor of thrombophilia in the western population, data about its prevalence among Indian patients is scanty. Recently, few reports have demonstrated that the PGM may exist in Indian patients [5, 6]. Therefore, we aimed to evaluate the prevalence of prothrombin gene mutations in (G20210A) among North Indian patients with venous or arterial thromboembolic events.
Materials and Methods
The patients with thromboembolic events referred to the coagulation laboratory of Department of Haematology of a tertiary centre in North India from July 2020 to June 2022 were prospectively included in the study. Additionally, the data of all patients with thromboembolic events in the archives of coagulation section of Hematology Department from January 2012 till June 2020 were retrieved and retrospectively reviewed. The study was approved by the Institutional Ethics Committee (No. INT/IEC/2022/SPL-236 dated 26.3.2022).
The thrombophilia work-up comprising PC, PS, AT and lupus anticoagulant (LA) levels were assayed using a fully automated coagulation analyzer (ACL TOP 500, Instrumentation laboratory, Bedford, MA, USA). Anticardiolipin (IgG, IgM) and anti-ß2GP1 (IgG, IgM) antibodies were performed by fully automated chemiluminescent assay analyser (ACUSTAR Instrumentation laboratory, Bedford, MA, USA). Deficiency of PC, PS or AT were confirmed by repeat testing after 1-month while positive antiphospholipid antibodies were corroborated after 3-months.
For the molecular genetics study, the genomic DNA was extracted from peripheral blood using ‘QIAamp® DNA Blood Midi kit’ (Qiagen, Germany) according to manufacturer’s instructions. Factor V Leiden (FVL) and prothrombin gene mutations (G20210A) were tested by polymerase chain reaction-restriction fragment length polymorphism (RFLP). Primer details are provided in Supplementary material. The positive results were confirmed using Sanger sequencing.
Data were analyzed using MedCalc® Statistical Software version 22.014 (MedCalc Software Ltd, Ostend, Belgium). Quantitative data have been expressed as mean standard deviation or median with interquartile range while qualitative data have been depicted as proportions and percentages.
Results
The study cohort comprised of 509 patients with thromboembolic events with male to female ratio of 1.61:1 (314 males and 195 females). The mean age of presentation in female and male patients were 33 and 32 years respectively. The most common clinical presentation was deep vein thrombosis (n = 208, 41%) while others had ischemic strokes (n = 178, 35%) cerebral venous sinus thrombosis (n = 72 (14%)), pulmonary thromboembolism, portal vein thrombosis, and arterial thrombosis. The demographic and clinicopathologic characteristics of the patients are shown in Table 1.
Table 1.
Demographic and clinicopathological profiles of whole cohort (n = 509)
| Demographic profile of patients with thromboembolism (n = 509) | |
| Mean age of presentation among males in years | 33 |
| Mean age of presentation among females in years | 32 |
| Male: Female | 1.6:1 |
| Clinical profile of patients with thromboembolism (n = 509) | |
| DVT only | 208 (41%) |
| DVT with PTE | 27 (5%) |
| Stroke | 178 (35%) |
| Cerebral vein thrombosis (CVT) and Cerebral sinus venous thrombosis (CSVT) | 72 (14%) |
| Portal vein thrombosis (PVT) and extra hepatic portal vein obstruction (EHPVO) | 13 (2.8%) |
| Arterial thrombosis | 11 (2.2%) |
In the whole cohort, confirmed thrombophilia risk factors were identified in 10.4% (n = 53) including hereditary risk factors in 42 (8.2%) patients and acquired risk factors were identified in 11 (2.1%) patients (Table 2). The most common inherited risk factor in our study cohort was FVL mutation [n = 31, (6%), heterozygous: 28, homozygous: 3] followed by isolated PS deficiency (n = 5, 1%). The PGM (PG20210A) was detected in only 2 (0.3%) patients in our cohort. These included a 25-year-old male from Chandigarh who presented with lower limb DVT and a 24-year-old male from Haryana who presented with acute ischemic stroke of the left frontal region. Both patients harbored heterozygous mutations.
Table 2.
Clinicopathological profile of confirmed inherited and acquired thrombophilia defect in the study population
| Clinical diagnosis | DVT | PTE | Stroke | CVT & CSVT | PVT and EHPVO | Arterial thrombosis | Total No. |
|---|---|---|---|---|---|---|---|
| Number of patients | 208 | 27 | 178 | 72 | 13 | 11 | 509 |
| Thrombophilia risk factors | 33 | 2 | 15 | 3 | 0 | 0 | 53 |
| Inherited thrombophilia risk factors-42 | |||||||
| Isolated PC deficiency | 2 | 0 | 1 | 0 | 0 | 0 | 3 |
| Isolated PS deficiency | 3 | 0 | 2 | 0 | 0 | 0 | 5 |
| Isolated AT deficiency | 1 | 0 | 0 | 0 | 0 | 0 | 1 |
| PG mutation | 1 | 0 | 1 | 0 | 0 | 0 | 2 |
| FVL | 20 | 0 | 8 | 3 | 0 | 0 | 31 |
| Acquired thrombophilia risk factors (Antiphospholipid antibodies)-11 | |||||||
| Isolated LA positive | 1 | 0 | 0 | 0 | 0 | 0 | 1 |
| Isolated ACA positive | 1 | 0 | 1 | 0 | 0 | 0 | 2 |
| Isolated β2GP1 positive | 0 | 0 | 1 | 0 | 0 | 0 | 1 |
| Multiple APLA positive | 4 | 2 | 1 | 0 | 0 | 0 | 7 |
Discussion
Globally, the incidence of VTE ranges from 0.75 to 2.7 per 1000 population [2]. These thromboembolic events are caused by an interplay between hereditary and acquired risk factors. However, there is paucity of Indian data on the various thrombophilia risk factors which necessitated the need for this study. In the present study a total of 509 patients were screened for thrombophilia workup with thrombophilic risk factor being confirmed in 53 (10.4%) patients. Among the various thrombophilia risk factors, FVL mutation is the commonest reported abnormality in India ranging from 3.7 to 28.5% which is in concordance with our observation of 8.5% (Table 3)[1, 5–12].
Table 3.
Indian studies on prevalence of thrombophilia risk factors in patients with venous thromboembolism or stroke
| Author (Year) | No. of VTE cases | Thrombophilia abnormalities | PC deficiency | PS deficiency | AT-III deficiency | APLA | FVL mutation | PGM |
|---|---|---|---|---|---|---|---|---|
| Patients with venous thromboembolism (VTE) | ||||||||
| Ghosh et al. (2001) [1] | 245 | 53 (21.6%) | 27 (9.8%) | 18 (7.34%) | 9 (3.67%) | Not done | 9 (3.67%) | 0 |
| Kumar et al. (2020) [13] | 50 | 20 (40%) | 3 (6%) | 5 (10%) | 3 (6%) | 4 (8%) | 5 (10%) | Not done |
| Rastogi et al. (2019) [14] | 100 | 56 (56%) | 3 | 4 | 2 | 31 | 16 | Not done |
| Rajpal et al. (2019) [15] | 100 | 32 (32%) | 0 | 9 | 0 | 11 | 6 | Not done |
| Sharma et al. (2022) [8] | 52 | 22 (44%) | 3 (5.77%) | 2 (3.84%) | 2 (3.84%) | 4 (7.69%) | 11 (21.15%) | Not done |
| Shafia et al. (2018) [5] | 250 | 24 (9.6%) | Not done | Not done | Not done | Not done | 17 (6.8%) | 7 (2.8%) |
| Present study | 235 | 35 (14.8%) | 2/98 (0.85%) | 3/98 (1.27%) | 1/98 (0.4%) | 8/173 (3.4%) | 20/235 (8.5%) | 1/235 (0.4%) |
| Patients with stroke or cerebral venous thrombosis (CVT) | ||||||||
| Ghosh et al (2001) [1] | 112 (CVT) | 27 (24.1%) | 12 (10.7%) | 10 (8.9%) | 3 (2.67%) | Not done | 2 (1.7%) | 0 |
| Chatterjee et al. (2013) [16] | 52 | 20 (38.4%) | 5 (9.6%) | 15 (28.8%) | 0 | 0 | Not done | 0 |
| Salomi et al. (2019) [6] | 392 | 5/392 (1.2%) | Not done | Not done | Not done | Not done | Not done | 5 (4/82 north Indian & 1/310 south Indian |
| Sharma et al. (2020) [9] | 180 (CVT) | 29 (16.1) | 7/148 (4.7%) | 4/147 (2.7%) | 2/146 (1.3%) | 9/174 (5%) | 7/180 (4%) | Not done |
| Narayan et al. (2021) [10] | 100 (25 CVT) | 56 (56%) | 26 (26%) | 46 (46%) | 41 (41%) | Not done | 0 | Not done |
| Gill et al. (2022) [12] | 75 | 9 (12%) | 4 (5.3%) | 3 (4%) | 2 (2.7) % | Not done | 1 (1.3%) | 0 |
| Present study | 250 (72 CVT) | 18 (7.2%) | 1/178 (0.5%) | 2/178 (1.1%) | 0 | 3/250 (1.2%) | 11/250 (4.4%) | 1/250 (0.4%) |
One of the striking observations about risk factors for thrombophilia is the stark variability in geographical distribution of prothrombin gene mutation. Linkage studies suggest that prothrombin gene mutation arose from a single founder event with subsequent geographical spread. The prevalence of prothrombin gene mutation may be as high as 6% among the general Caucasian population, it is extremely rare among those from Asian or African ancestry. PGM is a well-established risk factor for VTE and possibly arterial thrombosis the Western population and ranks second to only FVL mutation among the inherited thrombophilia risk factors [3, 4]. Traditionally, it was believed that PGM does not exist in India. However, two recent study reported the rare occurrence of prothrombin gene mutation among patients with VTE and stroke. A case–control study from Kashmir identified prothrombin gene mutation in 7 patients with VTE and none among control (p = 0.015) [5]. Another recent study reported PGM (G20210A) in 1.3% and 1.2% of patients with cerebral venous thrombosis and ischaemic stroke respectively [6]. Intriguingly, this study also found that PGM was more common among patients of North Indian ancestry (3.2% versus 0.8%; p = 0.026) [6]. In our cohort of North Indian patients, we identified only 2 (0.3%) patients with PGM (G20210A) who presented with VTE and ischaemic stroke. Our study adds to the growing body of evidence that PGM (G20210A) is uncommon but not absent among Indians and may predispose to thrombotic events.
We acknowledge the limitation of our study including relatively small sample size and lack of control group. Repeat work-up for antiphospholipid antibodies and level of protein C, protein S and antithrombin III could not be assessed in patients who had derangement of these parameters at index work-up. However, this does not hamper our inferences on prothrombin gene mutation analysis. Lastly various prothrombin gene variants apart from G20210A have been described in the literature confers the variable risk of thrombotic events. These alternate variants were not looked for in our study.
In conclusion, FVL mutation is the commonest risk factor among North Indian patients with thromboembolic events. PGM (G20210A) is rare but may occur in Indian patients. Further large multicentric studies are needed to better estimate the prevalence of this mutation and the conferred risk of thrombophilia in Indian patients. The role of PGM testing is still debatable due to its scarcity among north Indian population.
Supplementary Information
Below is the link to the electronic supplementary material.
Author Contributions
NK and PS contributed to the study conception and design. Material preparation, data collection and analysis were performed by PS, DR, VK, CH, HKS, JA, RD, AJ, KVM and NK. The first draft of the manuscript was written by DR and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding
None.
Declarations
Conflict of interest
None.
Ethical Approval
Approved by the Institutional Ethics Committee. All procedures performed were in accordance with the ethical standards of the institutional ethics committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Consent for Publication
Consent for publication was obtained for every individual person’s data included in the study.
Informed Consent
Informed consent was obtained from all individual participants included in the study.
Footnotes
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