Birendra Kumar Yadav

Molecular defects in ITGA2B and ITGB3 genes in patients with Glanzmann thrombasthenia

Summary.  Background: Glanzmann thrombasthenia (GT) is an autosomal recessive inherited platelet function defect that is characterized by reduction in, or absence of, platelet aggregation in response to multiple physiologic agonists. The defect is caused by mutations in the genes encoding ITGA2B or ITGB3. This results in qualitative or quantitative abnormalities of the platelet receptor, αIIb‐β3. Objectives: The aim of this study was to identify the mutations in GT patients and to correlate these with patient phenotype. Subjects and methods: A total of 45 unrelated patients with GT were enrolled in the present study to identify the causative molecular defects, and also to correlate their phenotype with their genotype. Platelet aggregation, flow cytometry, Western blotting, and mutation screening by conformation sensitive gel electrophoresis (CSGE) followed by sequencing were performed in all patients. Novel mutations were analyzed for penetrance in individual families. Results: A total of 22 novel mutations were identified in 45 unrelated GT patients. Mutations were identified in 36 of the 45 (80%) patients. Missense mutations were seen in most of the GT patients (59%). The remaining mutations were heterogeneous and were distributed throughout the length of the gene. Analysis of family members showed heterozygous mutations in all families. Conclusions: The severe type I GT was the most common subtype found in this study. Missense mutations were identified as the defects responsible for most GT patients. Carrier detection and genetic counseling in these families is a potentially effective alternative for decreasing the burden of severe type of GT.

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.

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.

Glanzmann's thrombasthenia in North Indians: Sub classification and carrier detection by flow cytometry

Thirty-three patients of Glanzmanns thrombasthenia (GT) and their families were assessed for the expression of αIIbβ3 on platelet surface, by flow cytometry, to determine the common subtypes in North Indians as well as to assess the carrier status in family members of GT patients. GT was diagnosed in patients with bleeding manifestations accompanied by absent/reduced platelet aggregation, secondary to adenosine-di-phosphate, adrenaline, arachidonic acid and collagen. Based on αIIbβ3 levels, 21 patients (64%) were classified as type I (as αIIbβ3 was absent), 4 patients (12%) as type II and 8 patients (24%) as type III. Eight out of 20 fathers, 10 out of 20 mothers and 20 out of 31 siblings were found to have reduced αIIbβ3 levels. Reduced αIIbβ3 expression was seen in 63% of parents and 65% of siblings. It is possible that low αIIbβ3 levels in family members may reflect their carrier status. It is postulated that flow cytometry estimation of αIIbβ3 in parents/siblings may detect carrier status in GT. It is also revealed that type I GT is the commonest subtype in North Indians.

Modulation of clinical phenotype of Glanzmann's thrombasthenia by thrombogenic mutations

BACKGROUND Glanzmanns thrombasthenia (GT) is an autosomal recessive bleeding disorder which is due to a defect in platelet aggregation in response to multiple physiological agonists. It has been demonstrated that the clinical phenotype of various diseases inherited in a classic Mendelian fashion can be modulated by a series of factors, inherited as well as acquired. METHODS A total of 45 GT patients were screened for the thrombogenic polymorphisms, i.e., FV Leiden (R506Q), Prothrombin G20210A, MTHFR C677T and HPA-1 by PCR/RFLP. RESULTS MTHFR C677T heterozygous was seen in 6 patients, FV Leiden heterozygous in one and Prothrombin G20210A gene variant in none. HPA-1 was seen in 3 patients of whom 1 was homozygous and 2 were heterozygous. CONCLUSION Thus the coinheritance of heterozygous FV Leiden alone or homozygous HPA 1b alone or the combined heterozygosity of MTHFR and HPA-1 were predicted to alter the clinical phenotype. Whereas the inheritance of heterozygous MTHFR alone or heterozygous HPA-1 alone did not altered the clinical phenotype significantly. Hence FV Leiden, MTHFR C677T polymorphism along with PLA-1 and homozygous HPA-1 were the probable ameliorating factor in GT mild phenotype.

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.