Arvind Meena

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.

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, χ2 = 10.303; odds ratio [OR] = 3.75) as well as allelic association (P = .0004, χ2 = 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.

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.

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.

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.

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.

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.

Inherited warfarin resistance in Indian patients: does it occur?

To the Editor: Warfarin resistance is a phenomenon that is observed in the administration of the anticoagulant warfarin to patients suffering from thrombotic disorders. This is diagnosed when patients do not respond to increasing dosages of warfarin, which would in most patients cause an adequate international normalized ratio (INR) elevation. Resistance to this drug is rare and may be due to both acquired and hereditary causes (1-3). In this study, we report the prevalence of warfarin resistance in deep vein thrombosis (DVT) cases in India; 139 patients of DVT were followed up in the hemostasis clinic Department of Haematology, All India Institute of Medical Sciences. Subjects comprised all of the DVT patients who maintained an INR between 2 to 3 by warfarin in excess of 8 mg/d or did not achieve an INR of 2 by doses of greater than 8 mg/d. Initially, they were started with 2 mg/d of warfarin, and the warfarin dose was gradually increased until an INR of 2 to 3 was achieved. A detailed clinical history was evaluated in these patients, taking special care of a vitamin K-rich diet (spinach and broccoli) and/or associated drug interactions such as antibiotics, amiodarone, statins, and anticonvulsants. One of our patients was receiving antituberculosis treatment, whereas the other was on a green leafy vegetable diet. In these patients, when the green leafy vegetables and antituberculosis treatment were stopped, INR became between 2 to 3 with a dose of 4 mg/d. The remaining 12 patients of the total 139 DVT (8.6%) continued to have warfarin resistance. All of these patients showed requirement of warfarin greater than 8 mg. Of these, 5 achieved an INR of between 2 to 3 when warfarin was increased to 10 mg/d; however, 5 did not achieve an INR of 2 with 12 mg/d of warfarin. One patient achieved an INR of 2.35 with 14 mg/d of warfarin, whereas 1 did not respond even at 15 mg/d. It is possible that these were cases of true warfarin resistance, which may have been due to hereditary warfarin resistance such as polymorphism in cytochrome P450CYP2C9 and P450CYP2A6 liver enzymes required for the oxidative metabolism of warfarin (4,5). A potential pharmacodynamic mechanism underlying warfarin resistance has been elucidated with the recent discovery of the warfarin target gene, which encodes vitamin K epoxide reductase complex (VKORC1). This complex recycles reduced vitamin K, which is essential for the posttranslational gamma-carboxylation of vitamin K-dependent clotting factors II (prothrombin),VII, IX, and X. Several rare mutations that lead to amino acid changes in the VKORC1 and a mutation in vitamin K epoxide reductase multiprotein complex (VKOR) (6-8) have been discovered in warfarin-resistant patients. Hereditary warfarin resistance has earlier been reported in different ethnic races. The haplotype frequencies of CYP2C9*1 and CYP2C9*2 in white patients were Clinical and Applied Thrombosis/Hemostasis Vol. 13, No. 3, July 2007 338-339 DOI: 10.1177/1076029607302446

Oxidation of some organic sulfides by benzyltrimethylammonium chlorobromate: a kinetic and mechanistic approach

The oxidation of 34 sulfides by benzyltrimethylammonium chlorobromate (BTMACB) resulted in formation of the corresponding sulfoxides. The reaction is first order with respect to both BTMACB and the sulfide. The solvent composition effect indicated that the rate of reaction increases with increase in the polarity of the medium. The reaction failed to induce the polymerization of acrylonitrile. There is no effect of added benzyltrimethylammonium chloride or potassium bromide. Chlorobromate ion is postulated as the reactive oxidizing species. The rates of oxidation of meta- and para-substituted phenyl methyl sulfides were correlated with Chartons LDR equation. The rates of oxidation of the ortho-compounds showed excellent correlation with the LDRS equation. The oxidation of meta-compounds exhibited a greater dependence on the field effect. The reaction is subject to steric inhibition when an ortho-substituent is present. The oxidation of alkyl phenyl sulfides is subject to both polar and steric effects of the alkyl groups. The polar reaction constants are negative, indicating an electron-deficient sulfur centre in the rate-determining step. A mechanism has been proposed involving formation of a halogenosulfonium cation in the slow step.