Clopidogrel resistance in North Indian patients of coronary artery disease and lack of its association with platelet ADP receptors P2Y1 and P2Y12 gene polymorphisms.

Aspirin and Clopidogrel are used in prophylaxis of patients undergoing percutaneous coronary intervention and long-term prevention of cardiovascular and cerebrovascular events. Clopidogrel resistance has been attributed to P2Y1 and P2Y12 adenosine diphosphate (ADP) receptor polymorphisms. This study enrolled 100 patients of coronary artery disease (CAD) who were on the maintenance dose of clopidogrel (75 mg OD) with or without aspirin. In addition, 10 received loading dose (300 mg) prior to percutaneous coronary intervention. Relevant clinical and drug history were elicited. ADP-induced platelet aggregation study and PCR-RFLP for P2Y1 (1622A > G) and P2Y12 (i-T744C) polymorphisms were performed. Two groups of controls were used for defining cut-off for platelet aggregation response. Follow-up data, wherever available was recorded. The most common pattern of aggregation response was disaggregation, either complete (46.4%) or partial (53.6%). A frequency of 13% clopidogrel non-responders and 19% semi-responders was found. All the cases were H1/H1 haplotype for P2Y12 gene polymorphism and 28 (29.2%) patients carried P2Y1 1622A > G (21(21.9%) AG and 7(7.3%) GG) gene polymorphism, the frequency being greater in clopidogrel responders compared to semi/non-responders but difference was not statistically significant. There was no statistically significant difference between responders and semi/non-responders in terms of the history of risk factor for CAD, concurrent atorvastatin use or past history of an acute vascular event. On follow up, the two patients who developed myocardial infarction/acute coronary syndromes (MI/ACS) were clopidogrel semi- and non-responder, respectively. Variability in clopidogrel response with 13% non-responders and 19% semi-responders was seen in this study with adverse outcome (MI/ACS) on follow up seen in two patients. Hence, poor response to clopidogrel may be related to increased likelihood of adverse long-term coronary event that may benefit from additional or alternative anti-platelet therapy. Clopidogrel resistance was not associated with ADP receptor P2Y1 and P2Y12 gene polymorphisms. Hence, it is postulated that clopidogrel resistance in CAD patients is multifactorial and not caused by single-gene polymorphisms.

Prevalence of factor V Leiden G1691A, MTHFR C677T, and prothrombin G20210A among Asian Indian sickle cell patients.

The prevalence of factor V (FV) Leiden G1691A, prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T mutations were investigated among 90 sickle trait, 61 sickle homozygous, 75 sickle beta thalassemia, and 15 HbSD Asian Indian sickle cell patients. In all, 297 healthy controls were evaluated to compare the polymorphism frequency. The prevalence of FV Leiden heterozygous G>A were significant in the group (P = .02), while PRT G20210A polymorphism was not seen among patients as well as controls. However, an increased frequency of the MTHFR 677 C>T genotype was seen among patients as well as controls, but this was not statistically significant (P = .13). This suggested a low impact of inherited hypercoagulability risk factors in the pathogenesis of sickle cell disease and/or its complications.

Plasminogen activator inhibitor-1 (PAI-1) gene 4G/5G promoter polymorphism is seen in higher frequency in the Indian patients with deep vein thrombosis.

INTRODUCTION: A number of prothrombotic and fibrinolytic disorders may lead to venous thrombosis. A 4G/5G polymorphism located in the promoter region of plasminogen activator inhibitor-1 (PAI-1) gene has been found to be commonly associated with the levels of PAI-1 and might be a risk factor for deep vein thrombosis (DVT). The aim of this study was to look for the potential association of this polymorphism with DVT in the Asian Indian population. MATERIAL AND METHODS: A total of 110 consecutive patients (M:F = 62:48) with idiopathic DVT and equal number of age- and sex-matched healthy controls were the study participants. All study participants were typed for the PAI-1 4G/ 5G polymorphism, factor V Leiden, factor V Hong Kong/Cambridge mutations, and HR2 haplotype. RESULT: The variant allele for the PAI-1 4G/5G polymorphism showed both genotypic (P = .0013, chi(2) = 10.303; odds ratio [OR] = 3.75) as well as allelic association (P = .0004, chi(2) = 12.273; OR = 1.99) with DVT. Factor V Leiden and factor V HR2 haplotype were observed in 10 (9.0%) and 13 (11.8%) patients, respectively. None of the study participants showed the factor V Hong Kong Cambridge mutations. CONCLUSION: Our study shows the association of 4G allele with DVT in Asian Indian population. The higher prevalence of 4G polymorphism in patients with DVT (compared with controls) seen in our study is in concordance with previous reports from the Caucasian population.

Prothrombotic factors and the risk of acute onset non-cardioembolic stroke in young Asian Indians.

INTRODUCTION: Several prothrombotic factors--both hereditary and acquired--are known to cause stroke. Commonly investigated causes are activated protein C resistance, factor V Leiden mutation, factor VIII levels, prothrombin 20210 G-to-A mutation, coagulation inhibitors such as proteins C and S, and antiphospholipid antibodies such as beta(2)-glycoprotein. OBJECTIVE: The literature on the prevalence of hematological defects pertaining to these variables in the Asian Indian stroke population is limited to a few isolated reports. In the current study we investigate the above-mentioned variables in 120 stroke patients (non-cardioembolic acute-onset stroke) and compare their status with the hematological profile of an equal number of healthy age- and sex-matched controls. MATERIAL AND METHODS: Plasma and blood leukocytes were collected from all patients and controls for performing hematological assays and molecular tests respectively. The mutations were detected using standard polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) procedures. Statistical analysis was done using SPSS version 12.0. RESULTS: Factor V Leiden (prevalence 8.3% in patients) and activated protein C resistance (prevalence 19.6% in patients) both showed a high degree of association (P<0.01) with the disease condition. However, contrary to common expectations, factor V Leiden was observed much less frequently in patients showing activated protein C resistance (10 out of 23; 43.4%) than is commonly observed in the Caucasian population (almost 90%). Post-acute-phase factor VIII levels were also found to be significantly associated with stroke: 125.6+21.1% number of profitable positions (NPP) for controls and 136.2+28.8% NPP for patients (P=0.001). CONCLUSION: factor V mutations, such as factor V Leiden, may be important risk factors for stroke in an Asian Indian population. Activated protein C resistance has a stronger association with stroke than factor V Leiden and may be caused by other factors such as elevated factor VIII levels in the Asian Indian population apart from factor V Leiden itself.

Homocystine levels, polymorphisms and the risk of ischemic stroke in young Asian Indians.

BACKGROUND: Homocysteine has been for a fairly long time been debated to be a risk factor for stroke. Opinions are divided as to whether raised levels of homocysteine seen in stroke patients are the cause or consequence of stroke. A large number of studies have been conducted in the Caucasian as well as on the Oriental population, which tend to suggest contradictory findings at many times. However, there have been no reports forthcoming from the Asian Indian population, which is a genetically different population than the previously studied populations. SUBJECTS AND METHODS: In our present study, we looked at homocysteine levels and four commonly seen polymorphisms of homocysteine metabolizing enzymes and their respective prevalence in 120 acute onset ischemic stroke patients compared with an equal number of age and gender matched healthy population. We also tested the influence of folic acid dosage (5 mg OD) on the levels of homocysteine and the allied vitamin supplements, vitamin B12 and folate in smaller groups selected from the larger group. RESULTS AND CONCLUSIONS: We found homocysteine levels to be significantly raised in the stroke population compared with healthy controls [patients: 12 micromol/L (range: 5.3-39.1 micromol/L), controls: 11.2 micromol/L (range: 6.2-14.2 micromol/L); P =0.001]. There was an almost total response to folic acid dosage as all hyperhomocysteinemic patients showed lowering of homocysteine levels in response to the dosage. The MTHFR 677 C > T polymorphisms showed association with both homocysteine levels as well as stroke (P < 0.001). Nutritional deficiency plays a dominant role in hyperhomocysteinemic conditions in our stroke population, however. Genetic determinants of homocysteine level may also have some part in determining hyperhomocysteinemic conditions in the Asian Indian populations.

Prothrombotic polymorphisms, mutations, and their association with pediatric non-cardioembolic stroke in Asian-Indian patients.

Genes involved in the hemostatic mechanism are logical candidate genes for association studies in prothrombotic conditions such as stroke. Since the underlying etiology in pediatric strokes is different than adults, looking for genetic causes would be the logical thing to do in the pediatric stroke population. Fifty-eight Asian-Indian stroke patients below 15 years of age and equal number of age- and sex-matched healthy controls were the subjects for the study. The subjects were screened for 13 polymorphisms and three mutations spread across seven different candidate genes involved in the hemostatic system. Of the 13 polymorphisms and three mutations studied, four polymorphisms, HPA-I, TAFI 147Ala>Thr, methylene tetrahydrofolate reductase (MTHFR) 677 C>T, and MTHFR 1298 A>C, showed significant association with the disease phenotype. MTHFR 677 C>T showed the strongest association and therefore may have a strong predisposing role for pediatric strokes. Gene-gene interaction studies showed a strong interaction between HPA-I and MTHFR 677 C>T polymorphism. The wild type of both these polymorphisms synergistically showed a strong protective effect [p < 0.0001, O.R: 10.06(4.26-23.71)]. Polymorphisms in HPA-I and MTHFR may have important predisposing roles in the development of pediatric stroke.

Thrombin activatable fibrinolysis inhibitor gene polymorphisms are associated with antigenic levels in the Asian-Indian population but may not be a risk for stroke.

Thrombin activatable fibrinolysis inhibitor [carboxypeptidase B2 (plasma), CPB2] is a basic carboxypeptidase, which inhibits fibrinolysis by cleaving the C-terminal lysine residues on plasmin-modified partially degraded fibrin. Plasma CPB2 concentrations have been reported to be under the control of numerous single nucleotide polymorphisms located in the regulatory and coding regions of the gene encoding CPB2 (CPB2). High functional CPB2 levels have been found to be associated with an increased risk for ischemic stroke. The present study investigated CPB2 antigen levels and associated CPB2 polymorphisms in an acute onset non-cardioembolic stroke population compared with an age- and sex-matched healthy control population. This is, to the best of our knowledge, the first such study done in an Asian Indian population. CPB2 antigen levels were significantly associated with the disease phenotype (P < 0.001) and with CPB2 polymorphisms (P < 0.001). The haplotypes generated on analysis of the genotypic data accounted for 21% of the natural variation in the CPB2 antigenic levels. However none of the haplotype combinations generated showed any association with disease phenotype and therefore could not explain for the difference in CPB2 antigen levels between cases and controls.

TAFI antigen level variability in young healthy Asian Indians; first report from Asia.

Thrombin Activatable Fibrinolytic Inhibitor (TAFI) is a plasma protein, which inhibits fibrinolysis by removing carboxyterminal lysine residues from partially degraded fibrin thereby decreasing plasminogen binding on its surface. In this study we have investigated the antigenic level variability (Inter and Intraindividual) of Thrombin Activatable Fibrinolysis Inhibitor in 120 healthy Asian Indians since no data on this is available regarding this population. TAFI antigen levels did not show a normal distribution in our population (p<0.001). Median TAFI antigen levels were found to be 11.683 microg/ml. It ranged from 33.9-202.5%of normal pool plasma (3.9-23.5 microg/ml). TAFI antigenic level showed high level of variability in the Indian population (coefficient of variation: 37.4%). TAFI antigenic levels were stable intraindividually (p=0.218).

Factor V Leiden: is it the chief contributor to activated protein C resistance in Asian-Indian patients with deep vein thrombosis?

BACKGROUND: Deep vein thrombosis is a condition, which has several acquired as well as genetic causes. One of the most common reasons for deep vein thrombosis is activated protein C resistance caused by Factor V Leiden. METHOD: We examined the risk posed by Factor V Leiden, Hong Kong/Cambridge and HR2 Haplotype mutations in 155 deep vein thrombosis patients and 120 healthy controls in the background of activated protein C resistance. RESULT: Thirty-one of our patients showed activated protein C resistance of which only 16 carried Factor V Leiden mutation which was a far lower number than what is usually seen in Caucasian population. Factor V Leiden mutation was significantly associated with the risk of deep vein thrombosis (Yates corrected p-value=0.002; 95%CI; odds ratio: 13.7). Factor V Hong Kong/Cambridge and HR2 Haplotype were not found to be associated with the risk of deep vein thrombosis. There is a possibility that Factor V HR2 Haplotype might also be associated with activated protein C resistance even in the absence of Factor V Leiden. CONCLUSIONS: Factor V Leiden mutation was seen to contribute far less towards activated protein C resistance in Asian-Indian deep vein thrombosis patients than what has been commonly observed in Caucasians.

Importance of investigating somatic and germline mutations in hemophilia A: a preliminary study from All India Institute of Medical Sciences, India.

BACKGROUND: Hemophilia A is a common hereditary bleeding disorder caused mainly by mutations in the Factor VIII (FVIII) gene, which results in defective or absent FVIII protein. Most of the causative mutations arise from the germ cells, which leads to either heterozygous or hemizygous state for the mutation in the next generation. Germline or somatic mosaic may result due to a de novo mutation during early embryogenesis. METHOD: We analyzed 14 families of Indian origin with Hemophilia A [sporadic and severe] for the presence of mosaic individuals by employing Allele Specific PCR, mutation enrichment experiment and sequencing. RESULT: Nine families had point mutations, 3 families had small deletions or insertions, 2 families had splice site mutations. The origin of the de novo mutation was assigned to the patients' mother in 8 families. For 4 families it was assigned to the maternal grandmother and to the maternal grandfather in 2 families. In a single family somatic mosaic was detected. CONCLUSION: The presence of somatic mosaic in families with sporadic Hemophilia A in India may confound risk estimation during genetic counseling.

Recurrent abortions in Asian Indians: no role of factor V Leiden Hong Kong/Cambridge mutation and MTHFR polymorphism.

Recurrent fetal loss is a frequent health problem. Data accumulated over the past few years have suggested a possible correlation between thrombophilia and fetal loss. Although a clear association has been established between fetal loss and certain thrombophilic states, such as antiphospholipid antibody syndromes, antithrombin deficiency, and combined defects, reports on the prevalence of inherited prothrombotic defects such as factor V Leiden mutation and methylene tetrahydrofolate reductase C677T polymorphism in fetal loss are contradictory. The prevalence of these 2 mutations in Asian Indians with recurrent fetal loss has not yet been studied. In light of this, the present study looked at the prevalence of these mutations in 85 patients with spontaneous recurrent abortion and 31 controls. The authors did not find any significant role of these mutations in the development of recurrent abortion.