| Abstract|| |
Background: There has been an alarming rise in the incidence of coronary artery disease (CAD) in India especially involving the age group of less than 45 years. In recent past, various studies focused on hemostatic aspects of CAD, but could not determine the significance of thrombophilic molecular marker in combination. The study was undertaken to investigate the association of thrombophilia related molecular markers in young patients with CAD. Materials and Methods: Thirty diagnosed patients with CAD of either sex under 40 years were included. Thirty healthy age and sex matched control subjects without evidence of CAD formed the control group. Detailed history and clinical examination findings were recorded. In addition to routine investigations, polymerase chain reaction (PCR) based molecular analysis for Factor V Leiden (FVL), methyltetrahydrofolate reductase (MTHFR) gene, tumor necrosis factor receptor 2 (TNFR2) gene, and prothrombin gene mutation were carried out. Results: The mean age (± SD) was 36.86 ± 3.90 years in the patients. Smoking was the most prevalent risk factor. FVL, MTHFR and TNFR2 gene mutation were seen in nine (30%) patient. Three patients had presence of more than one mutation. FVL, MTHFR and TNFR2 gene mutation was found in 4 (13.3%), 3 (10%), and 5 (16.6%) patients respectively. Prothrombin gene mutation was not seen in any of the subjects. There was no significant difference in lipid profile, fibrinogen levels and CRP among the patients with mutation and patients without mutation. Conclusion: Almost one-third of the cases were positive for the various mutations in the study and the presence of at-least one or the other risk factor adds on to the risk of future thrombosis. There is a need to demonstrate or document these mutations in a larger group further based upon ethnicity and geographic distribution.
Keywords: Coronary artery disease, Factor V Leiden , methyltetrahydrofolate reductase, prothrombin, thrombophilia, tumor necrosis factor receptor 2
|How to cite this article:|
Sherpa M, Sharma S, Avasthi R. Thrombophilic molecular markers in young patients (<40 years) with coronary artery disease. Indian J Pathol Microbiol 2012;55:343-6
|How to cite this URL:|
Sherpa M, Sharma S, Avasthi R. Thrombophilic molecular markers in young patients (<40 years) with coronary artery disease. Indian J Pathol Microbiol [serial online] 2012 [cited 2019 Nov 14];55:343-6. Available from: http://www.ijpmonline.org/text.asp?2012/55/3/343/101741
| Introduction|| |
Coronary artery disease (CAD) is looming large as a global epidemic afflicting both the developed as well as the developing countries. This modern epidemic as described by WHO has not spared the developing nation like India . The prevalence of CAD has increased both in urban and rural populations of India. CAD among Indians has been found to be more severe, manifesting at a younger age and following a malignant course.
CAD is a complex disorder resulting from many risk factors. The well known traditional risk factors include smoking, high level of cholesterols, apolipoprotein A, blood glucose and low levels of HDL cholesterol. The traditional risk factors have long been considered to play a major role in predisposition to CAD however recent insight into the human genetics using genome scan approaches have found novel genetic loci associated with CAD, which might provide an additional insight to genetic factors contributing to atherosclerosis and coronary events.
Different aspect of thrombophilic markers and their significance in young patients with CAD without traditional risk factor is still being investigated.
The thrombophilic markers in combination under our study include Factor V Leiden, methyltetrahydrofolate reductase (MTHFR) gene mutation, tumor necrosis factor receptor 2 (TNFR2) gene mutation and prothrombin gene mutation.
| Materials and Methods|| |
Thirty diagnosed patients of coronary artery disease (CAD) of either sex under 40 years and thirty healthy age and sex-matched control subjects without evidence of coronary artery disease (normal resting 12 lead ECG), and absence of angina, myocardial infarction (MI) or history of any vascular disease were included in the study.
The inclusion criteria included documentation of acute MI, based on history of retro-sternal chest pain, elevated cardiac enzymes (CPK-MB, Trop T) and pathological Q waves on ECG and or coronary angiographic evidence of severe (>70% stenosis of two or more coronary arteries). A detailed clinical history, including presentation, risk factors, family history, clinical examination, evidence for clinical diagnosis, and history of any drug intake were recorded in a predesigned proforma. Electrocardiographic findings were recorded along with angiographic findings wherever available.
Hemogram, blood sugar (fasting and postprandial), lipid profile, screening coagulation tests such as prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time were also carried out.
DNA was extracted from the 5 ml venous blood sample collected in K 2 EDTA in all cases and controls using phenol-chloroform extraction method followed by ethanol precipitation.  DNA analysis by PCR for Factor V Leiden (FVL), methyltetrahydrofolate reductase (MTHFR C677T) gene mutation, tumour necrosis factor receptor 2 (TNFR2) gene mutation and prothrombin G20210A polymorphism were carried out [Figure 1] and [Figure 2].
|Figure 1: Agarose gel electrophoresis: PCR reac on and RE digestion for TNFR-2 mutation|
Click here to view
|Figure 2: Agarose gel electrophoresis: PCR reaction product for prothrombin gene mutation|
Click here to view
| Results|| |
Present study comprises of 60 subjects including 30 patients and 30 controls. The demographic characteristic of cases and controls are shown in [Table 1]. When examined for traditional risk factors, smoking was found to be most prevalent in the patients with CAD followed by presence of hypertension, hypercholesterolemia and diabetes. Low levels of HDL cholesterol were found in both the groups. The incidence of dyslipidemia among the subjects under study is shown in [Table 2]. The mean fibrinogen levels in patients were 252.1 ± 114.8 mg/dl and 205.8 ± 52.4 mg/dl among control subjects. C-reactive protein (CRP) levels were found to be significantly high (P < 0.5) in (43.3%) cases.
|Table 1: Demographic characteristics and prevalence of risk factors for coronary heart disease among young MI and controls|
Click here to view
Nine of cases tested positive for the mutations, with TNFR2 gene mutation being most common (16.6%), followed by FVL (13.3%) and MTHFR gene mutation (10%) [Table 3]. The Prothrombin gene mutation was negative in all the patients. All the controls tested negative for the mutation under study. Presence of more than one molecular marker was seen in three (10%) patients, comprising of two males and one female patient. The cases positive for the mutation had presence of minimum of two other risk factors for CAD. There were no significant differences in total cholesterol, triglycerides and HDL levels among the patients with and without mutations (P = 0.18, 0.23, and 0.54, respectively).
| Discussion|| |
The relationship between the hemostatic system and arterial thrombosis is being actively investigated, but there are a limited number of such studies in India. CAD has not only reached alarming proportions in India but also the involvement of younger age group without the presence of conventional risk factors cannot be neglected. , The study was undertaken to with an objective to look for the presence the thrombophilic molecular markers in young CAD patients.
In our study, the mean age (± SD) was 36.86 ± 3.9 years in patient which falls within the definition of premature CAD.
Smoking as a risk factor was most prevalent being present in 21 (70%) of the patients. However, other traditional risk factors were present only in ten (33.3%) patients. Our findings of lower proportion of traditional risk factors in young CAD patients were similar to findings of other studies.
Fibrinogen levels are being evaluated extensively for its role in atherosclerosis in general and in premature CAD in particular. The fibrinogen levels have been found to be significantly elevated in MI patients as observed in Indian population. ,, High fibrinogen levels (>400 mg/dl) were seen in three (10%) of the patients, thus alarming its need to be investigated in association with CAD in a larger population based study.
High CRP value in patients with stable CAD remains a strong independent indicator for short and long term mortality, thirteen (43.3%) patients had a high CRP levels (CRP > 6 mg/l) Measurement of high-sensitivity CRP (hs-CRP) is a useful clinical marker of inflammation related to atherosclerosis but the shortcoming of our study was lack of measurement of hs-CRP.
The relationship between FVL and arterial disease is controversial with association seen in few and no association in other studies.,, Limited by the number of cases positive for mutation, an association of FVL with CAD could not be determined but their presence among the cases of CAD do suggest their role somewhere in the coronary event and cannot be neglected.
Elevated homocysteine is one of the well-known risk factor CAD. MTHFR gene mutation is commonest cause of mild hyperhomocysteinemia, this mutation was seen in three (10%) patients.
Alteration in the genes coding for molecules which are involved in the inflammation may modify the risk of CAD.  However, there is no convincing evidence in literature, which suggests the disease association of TNFR2 with CAD.  The CRP levels in five cases positive for TNFR2 was significant (P value < 0.5) to that of the cases without mutation. However, further studies are required to confirm this finding for the better understanding of the genetic basis of the inflammatory process leading to atherosclerosis and CAD.
The Prothrombin gene mutation was not found in any of the patients or control. Our findings were similar as observed in the previous Indian literature, suggesting the absence of the mutation in Indian population. ,,,
Among the three cases positive for more than one mutation, one presented with primary heart block and other underwent an angiography showing two vessel diseases.
Presence of a mutation along with multiple mutations within a limited number of cases leaves us with a question that can such hemostatic markers explain the rising incidence and the severity of CAD cases. Thus a larger population-based study investigating the presence of hemostatic markers and its role in arterial thrombosis needs to be carried out.
| Conclusion|| |
The presence of thrombophilic molecular marker in addition to the conventional risk factors adds on to the future risk of thrombosis. Thus there is a need to investigate for this thrombophilic molecular marker in larger population based on the geographic distribution and ethnicity.
| References|| |
|1.||Arruda VR, Annichma-Bizzaahi JM, Costa FF, Reitsma PH. Factor V Leiden (FVQ506) is common in a Brazilian population. Am J Haematol 1995;49:242-3. |
|2.||Reddy KS. Neglecting cardiovascular disease is unaffordable. Bulletin of WHO 2001;79:984-5. |
|3.||Reddy KS. Cardiovascular diseases in the developing countries: Dimensions, determinants, dynamics and directions for public health. Public Health Nutr 2002;51:231-7. |
|4.||Jose J, Selvakumar D, Selvakumar R, Kanagasapabathy L, Jayaseelan L. Plasma fibrinogen: An independent risk factor for ischaemic heart disease. Indian Heart J 1998;50:45-8. |
|5.||Gheye S, Lakshmi AV, Krishna TP, Krishnaswamy K. Fibrinogen and Homocysteine levels in coronary artery disease. Indian Heart J 1999;51:499-502. |
|6.||Deepa R, Velmurugan K, Saravanan G, Dwarkanath V, Agarwal S, Mohan V. Relationship of tissue plasminogen activator, plasminogen activator inhibitor-1 and fibrinogen with coronary artery disease in South Indian male subjects. J Assoc Physicians India 2002;50:901-6. |
|7.||Holm J, Zöller B, Berntorp E, Erhardt L, Dahlbäck B. Prevalence of factor V gene mutation amongst myocardial infarction patients and healthy controls is higher in Sweden than in other countries. J Intern Med 1996;239:221-6. |
|8.||Demarmels Biasiutti F, Merlo C, Furlan M, Sulzer I, Binder BR, Lämmle B. No association of APC resistance with myocardial infarction. Blood Coagul Fibrinolysis 1995;6:456-9. |
|9.||Chambers JC, Obeid OA, Refsum H, Ueland P, Hackett D, Hooper J, et al. Plasma homocysteine concentrations and risk of coronary heart disease in UK Indian Asian and European men. Lancet 2000;355:523-7. |
|10.||Andreotti F, Porto I, Crea F, Maseri A. Inflammatory gene polymorphism and ischaemic heart disease: Review of population association studies. Heart 2002;87:107-12. |
|11.||Sankar VH, Girisha KM, Gilmour A, Singh VP, Sinha N, Tewari S, et al. TNFR2 gene polymorphism in coronary artery disease. Indian J Med Sci 2005;59:104-8. |
|12.||Ghosh K, Shetty S, Madkaikar M, Pawar A, Nair S, Khare A, et al. Venous thromboembolism in young patients from western India: A study. Clin Appl Thromb Hemost 2001;7:158-65. |
|13.||Herrmann FH, Salazar-Sanchez L, Schroder W, Grimm R, Schuster G, Jimenez-Arce G, et al. Prevalence of molecular risk factors FV Leiden, FVHR2, FII 20210 G>A and MTHFR 677C>T in different populations and ethnic groups of Germany, Costa Rica and India. Int J Hum Genet 2001;1:33-9. |
|14.||Ghosh K, Khare A, Kulkarni B, Shetty S, Mohanty D. Geography too determines the causes of inherited thrombophilia. J Thromb Haemost 2004;2:363-4. |
|15.||Mohanty D, Shetty S, Ghosh K, Pawar A, Abraham P. Hereditary thrombophilia as a cause of Budd-Chiari syndrome: A study from Western India. Hepatology 2001;34:666-70. |
Department of Pathology, UCMS and GTB Hospital. Dilshad Garden, Delhi 110095
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]