A study of the PfNT3 in Plasmodium falciparum

Previous genetic studies demonstrated that survival and proliferation of Plasmodium falciparum parasites is dependent on salvage of essential purines from the host. Plasmodium falciparum, the causative agent of the most lethal form of human malaria lacks the enzymes required for de novo synthesis of purines. Analysis of the hypothetical nucleoside/nucleobase transporter protein, the gene product of PfNT3 (PF14_0662) gene in P. falciparum parasites was carried out by localisation, in view of a novel chemotherapeutic target. Immunoblotting, immunofluorescent and immunoelectron microscopic localization of PfNT3 was demonstrated using polyclonal antiserum in in vitro cultured Plasmodium falciparum parasites, propagated in human red blood cells. PfNT3 protein, the translated product of PfNT3 gene was detected in intraerythrocytic ring, trophozoite, and schizont stages. PfNT3 was localized primarily to the PPM (Parasite Plasma Membrane). The endogenous PfNT3 putative nucleoside transporter with the predominant location to the parasite plasma membrane may serve not only as routes for targeting of purine analogs/cytotoxic agents into the intracellular parasite but may also serve as drug targets. Being genome encoded the vital transporter protein can be prevented from expression by silencing of the gene, validating it to be a novel drug target.

Guidelines for screening, diagnosis and management of hemoglobinopathies.

The b‑thalassemias and sickle cell disorders are a major health burden in India. Diagnosis and management of these disorders both in adults and in newborns using appropriate approaches and uniform technology are important in different regions of a vast and diverse country as India. In view of a National Thalassemia Control Program to be launched soon, a need was felt for guidelines on whom to screen, cost‑effective technologies that are to be used as well as for establishing prenatal diagnosis programs in regional centers. Newborn screening for sickle cell disorders is in its infancy in India and uniform approaches need to be followed. Also, included are guidelines for monitoring and managing patients who are now growing older and need comprehensive care as well as management of complications of the disease.

A novel autologous stem cell procedure for the treatment of aplastic anaemia using reprogrammed mature adult cells: a pilot study.

Background & objectives: Aplastic anaemia is a life threatening rare bone marrow failure disorder. The underlying haematopoietic cellular deficit leads to haemorrhage, infection and severe anaemia. The treatment of choice for this haematological condition is allogeneic bone marrow transplantation from fully matched HLA sibling. Though this procedure is curative in the majority of young patients with aplastic anaemia, extending this benefit to older patients or those lacking a family donor remains a major challenge. Herein, the safety and efficacy of infusing autologous retrodifferentiated haematopoietic stem cells (RHSC) into four patients with aplastic anaemia without the use of any pre- or post-conditioning regimen including immunosuppression is described. Methods: Un-mobilized, mononuclear cells were harvested from four patients with acquired aplastic anaemia by aphaeresis. Mononuclear cells of patients were cultured with purified monoclonal antibody against the monomorphic regions of the beta chain of MHC class II antigens (Clone CR3/43) for 3 h, to obtain autologous RHSC. Autologous RHSC were washed and infused into the four patients without the use of any pre- or post-conditioning regimen. Thereafter, the efficacy (engraftment) of autologous RHSC was assessed in these patients. Results: Following single infusion of the autologous RHSC, two of the four patients with aplastic anaemia become transfusion independent for more than seven years. Karyotyping and G-banding analysis prior and post-procedure in all patients remained the same. Interpretation & conclusions: The findings of this pilot study demonstrated the functional utility of reprogrammed fully differentiated adult cells into pluripotent stem cells with extensive repopulation potentials in a human setting and without any pre- or post-conditioning regimen, including immunosuppression. This autologous approach of stem cell creation may broaden the curative potentials of stem cell therapy to a wider population of patients with aplastic anaemia, including many patients suffering from other haematological and non-haematological disorders.

Genetic counselling in tribals in India.

Genetic counselling in tribals unlike general population residing in cities and near villages is a difficult task due of their lower literacy and poor socio-economic status. However, sustained effort is essential with a close interaction in the local language, certain misbeliefs need to be removed gradually taking into account their socio-cultural background. The present communication deals with our experience in counselling for haemoglobinopathies during Neonatal Screening Programme undertaken for sickle cell disease in Kalahandi district of Orissa and Community Screening Programmes in primitive tribes of India in four States viz. Orissa, Gujarat, Tamil Nadu and Maharashtra. Counselling during neonatal screening programme was very well accepted demonstrating the benefit to the small babies as regards the morbidity. Premarital marriage counselling was also accepted by them. The success rate as followed up for 5 years is almost 50 per cent, the limitation being long follow up. Genetic counselling in these areas has to be continuous to achieve success and therefore the need for setting up of permanent centres in the tribal areas in India.

Clinical, hematologic and molecular variability of sickle cell-β thalassemia in western India.

BACKGROUND: Sickle cell-β thalassemia (HbS-β thalassemia) is a sickling disorder of varying severity, which results from compound heterozygosity for sickle cell trait and β thalassemia trait. The present study was undertaken to determine the genetic factors responsible for the clinical variability of HbS-β thalassemia patients from western India. MATERIALS AND METHODS: Twenty-one HbS-β thalassemia cases with variable clinical manifestations were investigated. The α and β globin gene clusters were studied by molecular analysis. RESULTS: Thirteen patients showed milder clinical presentation as against eight patients who had severe clinical manifestations. Four β thalassemia mutations were identified: IVS 1-5 (G→C), codon 15 (G→A), codon 30 (G→C) and codon 8/9 (+G). α thalassemia and XmnI polymorphism in homozygous condition (+/+) were found to be common among the milder cases. The βS chromosomes were linked to the typical Arab-Indian haplotype (#31). Framework (FW) linkage studies showed that four β thalassemia mutations were associated with different β globin gene frameworks. Linkage of codon 15 (G→A) mutation to FW2 is being observed for the first time. CONCLUSION: The phenotypic expression of HbS-β thalassemia is not uniformly mild and α thalassemia and XmnI polymorphism in homozygous condition (+/+) are additional genetic factors modulating the severity of the disease in the Indian subcontinent.

Hypercoagulable state in idiopathic ulcerative colitis: role of hyperhomocysteinemia and hyperfibrinogenemia.

BACKGROUND: Previous reports on hypercoagulable factors in inflammatory bowel diseases involve heterogeneous populations and patients on various medications. AIMS: To determine the frequency of thrombotic complications in ulcerative colitis (UC); to evaluate for hyperhomocysteinemia and its relationship to vitamin B12 and folate levels and methylene tetrahydrofolate reductase (MTHFR) mutation; and to evaluate for hyperfibrinogenemia and factor V Leiden mutation. METHODS: Eighty-six adult patients with UC were seen during the study period; 28 of them underwent blood tests and constituted the study population. Patients who received medications that affect these factors were among the 58 excluded. Tests were obtained at baseline and after 2 months during remission. Patients received folic acid in addition to treatment for UC. RESULTS: Vascular thrombotic events were noted in 4 patients during follow up. Hyperhomocysteinemia was detected in 11 (39.3%) patients (controls 15/100, p=0.007). Heterozygous state for MTHFR C677T mutation was found in 5 (17.9%) patients (controls: 0.2% homozygous, 13.6% heterozygous, p>0.05). Plasma homocysteine did not correlate with extent, severity or duration of disease, or with MTHFR C677T heterozygous state, but correlated with serum folic acid level (p=0.003) and BMI (p=0.03). With folate supplementation, homocysteine decreased significantly in patients who had hyperhomocysteinemia at baseline. Hyperfibrinogenemia was detected in 3 patients (none in 100 controls). Plasma fibrinogen was not affected by duration, extent or severity of UC and did not decrease with remission of disease. Only one patient had heterozygous factor V Leiden mutation. CONCLUSION: Vascular thrombosis occurred in less than a fifth of the UC population studied. Hyperhomocysteinemia reversible by folate supplementation and hyperfibrinogenemia were observed, but their contribution and that of factor V Leiden mutation appear to be insignificant.

Potential of commercial anti-D reagents in the identification of partial D variants in Indian population.

BACKGROUND & OBJECTIVE: As partial D variants are of clinical importance in transfusion medicine, the present study aims to determine the efficiency of commercial anti-D reagents to identify partial D variants. METHODS: Forty two samples of known partial D identified in the Indian population were tested with seven commercial monoclonal anti-D reagents. RESULTS: Most of the monoclonal anti-D reagents gave strong positive reactions (24 to 59%) to weak positive (28 to 47%) with partial D cells. Polyclonal anti-D detected all partial D variants as RhD positive, though reacting weakly with the majority (83%) of them. All the seven commercial monoclonal anti-D reagents detected some variants as D negative. Analysis of pairs of these reagents showed that the combinations of reagents 1 & 2 and 1 & 6 could detect all partial D variants as RhD positive and hence can be used for donor testing. INTERPRETATION & CONCLUSION: Findings of our study showed that none of the monoclonal reagents when used individually could detect all partial D variants. A combination of two suitable anti-D reagents are necessary to detect maximum number of partial D variants.

Red cell pyruvate kinase deficiency in neonatal jaundice cases in India.

OBJECTIVE: Pyruvate Kinase (PK) deficiency is the most common enzymopathy of the glycolytic pathway in erythrocytes. It constitutes one of the common causes of hereditary non-spherocytic hemolytic anemia. The aim of this study was to screen newborns in India for pyruvate kinase (PK) deficiency in relation to unconjugated hyperbilirubinemia. METHODS: Laboratory investigations done included complete blood counts, reticulocyte counts, direct and indirect bilirubin, assay of G6PD and PK activity, ATP and 2,3 DPG levels. All variables were studied in 50-cord blood samples from normal deliveries and 218 neonates with hyperbilirubinemia. RESULTS: 7 of the 218 cases of neonatal jaundice were PK deficient with 30-40% reduction in PK activity. These cases also had a 3-4-fold increase in 2,3 DPG:ATP ratios, which is one of the additional indicators for PK deficiency. Six of the 7 infants had a severe clinical course. CONCLUSION: This study shows that the prevalence of PK deficiency in Indian neonatal jaundice cases is 3.21%, which is relatively high. This emphasizes the need for screening neonatal hyperbilirubinemia cases in India for PK deficiency.

Prenatal diagnosis of von Willebrand disease in a family.

We report the successful prenatal diagnosis of von Willebrand disease (VWD) in a family with type 3 severe VWD by the indirect method of gene tracking using polymorphic markers of intron 40 of the von Willebrand factor (VWF) gene. The couple had a daughter diagnosed to have type 3 VWD. Chorionic villus sampling (CVS) was done in the eleventh week of gestation of a subsequent pregnancy. The 3 VNTR polymorphic markers VWF1, VWF2 and VWF3 of intron 40 of the VWF gene were used for linkage studies. DNA in the affected VWD patient, the father and mother as well as in the CVS using VWF1 and VWF3 polymorphic markers revealed that the foetus was affected. The family chose to abort the foetus. In a subsequent pregnancy, similar investigation revealed a normal foetus. Prenatal diagnosis in families with a diagnosed case of VWD can be used to determine the status of the foetus. The technique is inexpensive.

Prevalence and spectrum of von Willebrand disease from western India.

BACKGROUND AND OBJECTIVE: von Willebrand disease (VWD) is one of the most common inherited bleeding disorders in the west. Limited studies from India showed a prevalence of approximately 10 per cent of VWD among the cases with hereditary bleeding disorders. VWD remains an underdiagnosed entity in India. The prevalence of different subtypes of VWD is also not known which is essential for a proper management of these cases. The present study was thus undertaken to know the prevalence of VWD and its various subtypes in the western part of our country. METHODS: A total of 796 consecutive patients presented with various bleeding manifestations were analysed. The initial screening and confirmation tests for the diagnosis of VWD included bleeding time (BT), screening coagulation tests i.e., prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), factor VIII: C assay, ristocetin-induced platelet aggregation (RIPA) and VWF antigen (VWF:Ag) estimations. VWF multimer analysis, ristocetin cofactor activity (RCOF), VWF collagen binding assay (VWF: CBA), factor VIII : VWF binding assay were also done to classify and subtype these cases. RESULTS: The patients were subtyped as per the International Society of Thrombosis and Haemostasis (ISTH) criteria. Of the 796 patients screened, 58 were diagnosed as VWD. Of the 15 families with a positive family history of bleeding, 26 additional cases were diagnosed as VWD. Majority of the patients were type 3 (59.5%) with severe clinical manifestations, about 18 per cent of type 1 VWD patients were detected in this group while the prevalence of the qualitative variants of VWD i.e., type 2 VWD was found to be 19 per cent and the prevalence of various subtypes were type 2A (9.52%), type 2B (4.76%), type 2M (1.2%), type 2N (3.6%). INTERPRETATION AND CONCLUSION: The high prevalence of type 3 and a low prevalence of type 1 VWD which is in contrast to the western reports, suggests the low awareness of the disease as also the underdiagnosis of the mild cases in our country.

von Willebrand disease: a laboratory approach.

von Willebrand disease (VWD) is a common inherited bleeding disorder. Accurate diagnosis and classification of VWD is crucial for clinical management. A detailed clinical history, including that of bleeding, is required. A family and drug history are also important. Genetic factors such as blood group, and environmental factors such as stress, trauma, pregnancy and inflammation should also be considered. The age, ethnic group and hormonal status could also affect the von Willebrand factor (VWF) levels. No single test is robust enough to detect all variants of VWD. In view of the heterogeneity of the disease and limitations in assays, a battery of tests should be performed before a final diagnosis can be reached. These include the screening coagulation tests, factor VIII:C assay, VWF antigen assay, assessment of functional VWF which includes VWF ristocetin cofactor assays, VWF collagen binding assay, ristocetin-induced platelet aggregation and VWF multimer analysis. The newer ELISA techniques based on VWF binding with factor VIII glycoprotein (Gp) 1b and cerebrosides have also helped in determining certain unusual forms of VWD. The advent of new systems such as platelet function analysers (PFA), thromboelastography (TEG) and clot signature analysers (CSA), which are designed to assess either the primary platelet function or as a global haemostasis screen, have facilitated and simplified the diagnosis. However, few centres all over the world can perform all these expensive tests to provide a final diagnosis of VWD. We reviewed the laboratory investigations required for a diagnosis of VWD. Apart from congenital VWD, the possibility of acquired VWD should be considered in those with a negative past history of bleeding or in the presence of an underlying disease.

Combination of thrombophilia markers in acute myocardial infarction of the young.

BACKGROUND: The pathogenesis of arterial thrombotic disease involves multiple genetic and environmental factors related to atherosclerosis and thrombosis. But, there have been very few studies in India which have investigated some of the thrombophilia markers. AIM: To look for combined thrombophilia in MI patients. SETTINGS AND DESIGN: One hundred twenty patients of myocardial infarction (age below 40 yrs.) were recruited 8-10 weeks after stabilization. Hundred age and sex-matched healthy controls were also recruited in the present study. METHODS AND MATERIAL: Following thrombophilia markers were screened in these patients--plasma fibrinogen, protein C, protein S, antithrombin III, factor V Leiden, PT G20210A polymorphism, MTHFR C677T, homocysteine, fibrinogen b448 Arg/Lys polymorphism and CBS T833C mutation. STATISTICAL ANALYSIS: Statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 10.0, SPSS Inc., Chicago, USA. RESULTS AND CONCLUSION: Elevated fibrinogen levels, homocysteine (p< 0.001 and homocysteine with odds ratio 6.26) and factor V Leiden (p=0.038) were independently associated with MI in our patients. A total of 37 patients (42.5%) had the presence of more than one thrombophilia markers in combination. Out of these, 10 had the presence of three markers in combination and 1 had five thrombophilia markers in combination. Only 2 controls had prothrombotic markers in combination. Combined prothrombotic risk factors were significant in cases in comparison to controls (p< 0.001). Further larger studies on a nationwide basis recruiting a large number of young MI patients should be done to substantiate these findings.

Glucose-6-phosphate dehydrogenase deficiency in India.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the commonest red cell enzymopathy in humans and has an X-linked inheritance. It has been reported from India more than 30 years ago and the prevalence varies from 0-27% in different caste, ethnic and linguistic groups. The major clinical manifestations are drug induced hemolytic anemia, neonatal jaundice and chronic non-spherocytic hemolytic anemia. Individuals with G6PD deficiency have a selective advantage against falciparum malaria. Thirteen biochemically characterized variants have been reported from India. At the molecular level, G6PD Mediterranean is the most common deficient variant in the caste groups whereas, G6PD Orissa is more prevalent among the tribal of India. The third common variant seen in India is G6PD Kerala-Kalyan.