Re-evaluation of thrombophilia testing in clinical practice: Insights from an Indian cohort

INTRODUCTION

Thrombophilia is characterized by an increased susceptibility to developing blood clots in the veins and arteries. Thrombosis can be caused by a genetic or acquired abnormality in the clotting system, resulting in an imbalance between pro-coagulant and anticoagulant factors in the blood.^[1] The inherited factors mainly include factor V Leiden, prothrombin gene mutation, protein C deficiency, protein S deficiency, and anti-thrombin deficiency.^[2] Later, genetic variants in genes encoding coagulation factors – Factor V and prothrombin (Factor II) were also classified as significant mutations that cause venous thromboembolism (VTE).^[3] Other factors such as elevated Factor VIII, elevated Factor IX, hyperhomocystenemia, dysfibrinogenemia, etc have also been described in recent years.

Acquired thrombophilia includes external or environmental factors that increase an individual’s predisposition to form abnormal blood clots. Trauma, malignancy, use of contraceptives, hormone replacement therapy, antiphospholipid syndrome, and heparin-induced thrombocytopenia are some of the causes of acquired thrombophilia.^[4] Thrombophilia can increase the risk of developing VTE, such as deep vein thrombosis (DVT), pulmonary embolism (PE), stroke, heart attack, and other cardiovascular diseases.

According to the Centre for Disease Control and Prevention, VTE is one of the most common causes of death and affects almost 9,00,000 people in the US, and increases the risk of recurrent thrombotic events in almost 33% of affected patients.^[2,5]

VTE is the most common clinical feature that is observed due to inherited mutations and serves as a major health issue worldwide. DVT and PE are the commonly detected VTE events. VTE is also observed in other areas of venous circulation, such as the cerebral vein, portal vein, mesenteric vein, and renal vein.^[6] Arterial thrombosis is observed in the arteries supplying blood to the brain, which can lead to a brain stroke. DVT often occurs in the veins of the lower limbs and can also cause complications if the thrombus from the deep vein dislodges and travels to the pulmonary circulation. This current study followed the American Society of Hematology (ASH),^[7] the British Society of Hematology (BSH),^[8] and the American College of Chest Physicians (ACCP)^[9] guidelines for thrombophilia testing. This study was designed to assess the role of thrombophilia testing in relation to clinical diagnosis and treatment.

MATERIAL AND METHODS

After obtaining clearance from the ethical committee, the retrospective cross-sectional study was conducted at Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute. The study involved 292 cases that underwent thrombophilia mutation panel testing using reverse transcription polymerase chain reaction (RT-PCR) over 3 years in a tertiary healthcare hospital located in Mumbai, India. DNA samples were tested for mutations in Factor V, Factor II (prothrombin gene), and Methylenetetrahydrofolate Reductase (MTHER) genes using real-time PCR.

RESULTS

In the present study, the mean age of positive cases was 41.64 ± 16.15 years. Out of a total of 126 patients, 80 were male, and 46 were female [Table 1].

Figure 1a depicts that Factor V mutation is commonly associated with increased risk of thrombosis. Among 97 (33%) patients tested, only 11 (8.7%) were positive, indicating a relatively low detection rate within this cohort.

Close

Figure 1:

(a) Incidence of patients who tested positive for Factor V, Prothrombin, and MTHFR diagnosed with thrombosis. (b) Spectrum of single and double MTHFR positive mutations. MTHER: Methylenetetrahydrofolate reductase

Export to PPT

Prothrombin Factor (Factor II) Mutation showed a very low detection rate (0.8%) among the small 1 case tested. This may suggest a lower prevalence or possible under-representation in the tested population and a lack of proper guidelines for testing.

MTHFR mutations were tested in 159 (54.5%) of the cases, with a high detection rate of 114 (71.7%), suggesting this mutation is common in the tested group. The A1298C variant (76; 66%) was more common than the C677T (22;19.2%) variant, with both mutations present (16;14%), which can be clinically significant depending on the patient’s symptoms and biochemical findings. The presence of C677T mutation, either heterozygosity (14 cases) or homozygosity (8 cases) may be particularly relevant, as this genotype is often associated with elevated homocysteine levels and increased risk for vascular issues [Figure 1b].

Table 2 illustrates clinical presentation in the majority of patients that carry the heterozygous form of the C677T mutation. However, a significant portion (over one-third) is homozygous. Males made up the majority (72.7%) of cases with MTHFR C677T mutations in this group. Cerebral venous thrombosis (31.8%) was the most frequently observed venous complication. Other thrombotic events such as DVT (9.09%) and PE (13.6%), were also notable. The presence of elevated homocysteine in one patient (4.54%) supported the known metabolic impact of the MTHFR C677T mutation, especially in homozygous individuals.

Arterial events, particularly acute ischemia (18.1%), were present in a significant proportion of cases. Stroke presentations (9.09%), reflect the vascular risk associated with MTHFR C677T, especially in homozygous carriers. One case of multiple myeloma (4.54%) was noted.

The majority of individuals with the A1298C mutation were heterozygous. Males represented a higher proportion (60.5%) of cases. Venous thrombotic complications were the most common clinical presentation. Both PE and cerebral venous thrombosis were seen in 17.1% of patients, indicating a strong association between A1298C and venous thrombosis risk. Other serious conditions, such as portal vein thrombosis (3.94%) and Budd–Chiari syndrome (1.31%), further highlighted the venous thrombotic potential of this mutation. Arterial ischemic conditions also contributed significantly, particularly general ischemia (11.8%) and strokes (1.31%). Combined Venous and Arterial cases indicated systemic thrombotic tendencies involving both arterial and venous systems, further underscoring the importance of comprehensive vascular risk evaluation in patients with MTHFR mutations.

All patients were heterozygous for Factor V Leiden. Females were more represented (63.6%) in this group. The majority of clinical events (81.8%) were venous in nature. DVT (54.5%) was the most common manifestation, present in over half the patients. Cerebral venous thrombosis (18.1%) and PE (9.09%) also occurred, reflecteing the mutation’s systemic thrombotic risk. The data showed a single case of DVT (100%) associated with Factor II, in a male patient.

87.5% of cases were associated with venous conditions, and 12.5% of cases were associated with arterial conditions. Among individuals with MTHFR C677T and A1298T mutations, venous complications were significantly more common than arterial ones [Figure 1b]. This may indicate a stronger association of these mutations with venous thrombotic events compared to arterial events [Figure 2 and Table 3].

Close

Figure 2:

Co-mutational spectrum of MTHFR C677T and A1298T mutations. MTHER: Methylenetetrahydrofolate reductase

Export to PPT

DISCUSSION

Numerous studies have been conducted to illustrate the significant variations in the prevalence of genes associated with VTE. The primary genetic cause of VTE is polymorphisms in the Factor II (G20210A), Factor V (R506Q, H1299R), and MTHFR (C677T, A1298C) loci.^[10]

BSH has recommended testing in patients with arterial thrombosis and Ischemic stroke, while ASH does not recommend the same.^[7,8] In the present study, the age group 31–40 showed the highest burden of thrombophilia mutations, followed closely by the 21–30 and 41–50 groups. The testing and mutation incidence decrease significantly after 50 years of age. The findings highlighted the importance of screening in younger to middle-aged adults, particularly when clinical suspicion is high. The high positive-to-tested ratio in most groups suggested that testing is effectively identifying at-risk individuals. A similar study was conducted by Patel et al.^[11] reported the mean age of the study population was 38.6 ± 14.6 years. Kumari et al.^[12] found the mean age of patients was 32 ± 3.96 years. The high positivity rate indicated that at-risk individuals can be effectively identified through targeted genetic testing that is customized to their early symptoms. Early detection is essential for the purpose of guiding clinical decisions and reducing the likelihood of future thrombotic events by prolonging the duration of anticoagulation.

The occurrences of VTE events and different mutations were more in males than in females (63.4% males, 36.5% females). Patel et al.^[11] reported the male predominance in their study. Marco Cattaneo et al.^[13] also found male prevalence in their study. Although the precise reasons for the increased risk of thrombophilia mutations in males remain unclear, several factors have suggested a potential correlation to lifestyle factors such as smoking, hypercholesteremia, trauma, or surgery. Exposure to oral contraceptives or pregnancy in females can be defined factors. The probability of thrombosis is influenced by environmental variables, and ageing in addition to the cumulative effects of hereditary risk factors.^[14]

ACCP considers inherited thrombophilia testing, like factor V, when evaluating the risk of reccurrence after the first unprovoked VTE.^[9] All 11 patients (11.3%) with factor V (R506Q) mutation had heterozygous mutations and mainly presented with DVT (54.5%) and cerebral venous thrombosis (18.1%). Other clinical indications such as PE (9.09%), stroke (9.09%), and SMA thrombosis (9.09%) were also observed in factor V leiden (FVL)-affected patients [Table 4]. In a comparable study, Kalpage et al.^[15] found that the FV c.1691G>A mutation was substantially associated with venous thrombosis in contrast to arterial thrombosis. This was consistent with the findings of previous research conducted by Leonard^[16] and Catto et al.^[17] on the FV mutation, which demonstrated a predominance in venous thrombosis. ACCP has defined that heterozygous factor V has 5-fold increased risk, while there is a 10-fold increase in homozygous patients. In addition, our patient cohort did not contain any homozygous FVL mutations. Our patient cohort did not undergo familial screening.

ACCP does not recommend prolongation of anticoagulation therapy in positive factor II cases and testing at initial thrombotic events. Only one patient was found to be positive for Factor II in thrombophilia screening, with a clinical diagnosis of DVT (100%) in a male patient. Similar results were observed by research conducted by Ferraresi et al.^[18] and Koksal et al.^[19] According to their studies, the FII mutation was seen in association with venous thrombosis more than arterial thrombosis. Another study conducted by Kalpage et al.^[15] reported contrary results. Arterial thrombosis was observed in FII-positive patients in their cohort. Consequently, a greater number of patients must be tested to obtain conclusive feedback.

The most prevalent mutation in our Indian cohort group was MTHFR (C667T and A1298C), which was present in up to 71.69% of patients tested in the thrombophilia panel. This revelation questions the guidelines laid out for testing these mutations. The finding also emphasizes testing for the MTHFR gene if all the other causes of thrombophilia are found to be negative. Nevertheless, the literature on MTHFR mutation studies continues to raise questions about the genetic testing that is conducted to detect thrombosis and the association of MTHFR mutations with it. However, certain studies have established a correlation between the presence of an MTHFR mutation and an elevated risk of recurrent miscarriages and VTE, which includes PE and DVT.^[20]

MTHFR is an enzyme that is essential for the metabolism of folate, which involves the conversion of homocysteine to methionine. The C677T mutation in MTHFR can result in an accumulation of homocysteine, a thermolabile variant of the gene which causes reduced enzyme activity, as per the ACMG practice guideline. The presence of C677T heterozygosity and homozygosity (14:8) was found in these cases. Hyperhomocysteinemiais frequently linked to MTHFR homozygous C667T gene mutations, leading to damage of blood vessels.^[21] Venous thrombosis has been identified as a risk factor for mild to moderate hyperhomocysteinemia, which has also been linked to other cardiovascular diseases, including coronary artery disease. Hyperhomocysteinemia is a multifactorial condition that is the result of a combination of genetic, physiologic, and environmental factors.^[22] Cerebral venous thrombosis (18.1%) was the most frequently observed venous complication. Other thrombotic events, such as DVT (9.09%) and PE (13.6%), were also notable. Arterial events, particularly acute ischemia (13.6%), were present in a significant proportion of cases. Stroke presentations (4.54%), posterior circulation stroke (4.54%), reflected the vascular risk associated with MTHFR C677T. According to a study conducted by Moll and Varga,^[22,23] the 677C>T heterozygous mutation is the most prevalent form of MTHFR mutation, resulting in reduced enzyme activity. The results of this investigation were inconsistent with our investigation. The significant prevalence of C677T MTHFR mutations was also reported by Cattaneo et al.^[13] In addition, they noticed a significant proportion of DVT patients who were homozygous for the C677T mutation in the MTHFR gene.

The majority of individuals with the A1298C mutation were heterozygous. Both PE and cerebral venous thrombosis were seen in 17.1% of patients, indicating a strong association between A1298C and venous thrombosis risk. Other serious conditions, such as portal vein thrombosis (3.94%) and Budd–Chiari syndrome (1.31%), further highlighted the venous thrombotic potential of this mutation. Arterial ischemic conditions also contributed significantly, particularly general ischemia (6.57%) and strokes (1.31%). Nair et al.^[24] conducted a meta-analysis on the Indian population, which also revealed an association between A1298C MTHFR mutations and recurrent pregnancy loss in women. Healthy Lebanese and Syrian populations have been identified as having some of the highest frequencies of MTHFR mutations (C677T and A1298C) in a comparable study conducted by Assaf et al.^[25] In addition, Kumari et al.^[12] also reported a high frequency of MTHFR mutations in their study subjects. In the current research, the A1298C variant was more prevalent than the C677T variant.

Compound heterozygosity, which is the presence of one copy of each variant, was observed in 14.0% of patients with mutations. The clinical significance of this phenomenon is contingent upon the patient’s symptoms and biochemical findings. Venous conditions were associated with 87.5% of cases, while arterial conditions were associated with 12.5% of cases with MTHFR C677T and A1298T mutations. Venous complications were substantially more prevalent than arterial complications among individuals with MTHFR C677T and A1298T mutations. This may suggest a more robust correlation between these mutations and venous thrombotic events than with arterial events. Certain severe complications are associated with these synergistic mutations, as evidenced by the distribution of thrombotic events across various regions and the presence of double mutations.

BSH 2022 has recommended testing for arterial embolic stroke, rare anatomical sites such as cerebral venous thrombosis, and unusual locations such as splanchnic, renal, and hepatic veins.^[8] In our study, the testing was done for the above clinical conditions and found to be positive in 71.7% of cases. The limitations of the study were the lack of availability of family history, smoking history, history of APLA syndrome, biochemical parameters such as homocysteine levels, and lack of awareness of the genetic association of thrombophilia.

CONCLUSION

A total of 126 cases were identified as positive for either Factor V (11; 8.7%), Factor II (1; 0.8%), or MTHFR (114; 90%). The highest incidence of positive cases was seen in the age group of 30–40 years. MTHFR (71%) was the most frequently detected mutation in individuals and was found to be significantly associated with thrombosis in the Indian scenario. The risk prediction of MTHFR mutations for thrombosis needs to be defined for MTHFR C667T mutations in the homozygous state in association with homocysteine levels. Therefore, the study concludes its routine screenings for MTHFR in at risk patients after the other genetic markers are concluded out. This can assist in determining the relative risk of MTHFR mutation presence, thereby enabling the accurate management of patients with appropriate anticoagulation therapy if needed.