Biomedical subjects
Publications and source records attributed to Kanjaksha Ghosh.
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During the last 10 years (1995-2005) we have offered diagnosis in 438 families out of 502 families referred, by chorionic villus sampling procedure between 10 and 12 weeks of pregnancy in severe haemophilia A and B families. Sixty four families could not be offered a diagnosis in the first trimester either due to non-informativeness with the available techniques or due to the absence of affected members in the family and were subsequently offered diagnosis in the second trimester by direct analysis of factor VIII/IX clotting activity and antigen in the fetal blood samples. For first-trimester diagnosis in the chorionic villus samples, we have used both direct and indirect methods, that is, intron 22 and 1 inversions in the factor VIII gene, a multiplex PCR for the detection of gross deletions in the factor IX gene and RFLP analysis using a battery of markers within and outside the factor VIII/IX gene. The informativeness of all these techniques was found to be 92.21% in case of haemophilia A and 83.91% in case of haemophilia B. We followed up 122 children out of 326 (diagnosed unaffected prenatally) after birth and the diagnosis was adequately reconfirmed by both factor assays and by DNA analysis. Only one case of misdiagnosis was detected so far (0.22%), where the diagnosis was based only on the extragenic marker of the factor VIII gene.
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von Willebrand disease (VWD) is caused by qualitative or quantitative defects, or both, of the von Willebrand factor, a multimeric, high-molecular-weight glycoprotein. Type 2N VWD differs from other subtypes in that it shows normal primary haemostasis as all the routine tests performed for the diagnosis of VWD are normal. They present with defective coagulation, due to a moderate or severe deficiency of factor VIII, and consequently often they are misdiagnosed as either mild haemophilia A or type 1 VWD. Thus the actual prevalence of this subtype is not known in many of the populations. The differential diagnosis of this subtype is mainly based on the in-vitro capacity of plasma von Willebrand factor to bind exogenous factor VIII by immunoassay. However, most of the routine laboratories in developing countries do not have the testing facilities for type 2N VWD, mainly because of the cost of the reagents involved and the complexity of various assays reported. We report here the prevalence of type 2N VWD in an Indian population by developing a simple, not too expensive, user-friendly enzyme-linked immunosorbent assay technique and compared it with the reported technique of the factor VIII binding assay. Nine out of 97 VWD patients (10%) were found to be type 2N VWD using both the reported assay and the in-house ELISA; thus indicating that the prevalence of type 2N VWD is high in our population. The simple ELISA technique described here should be available in all routine laboratories in developing countries.
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The efficacy of the two von Willebrand factor (vWF) intron 40 variable number of tandem repeat (VNTR) markers, vWF1 and vWF2, in the genetic diagnosis of von Willebrand disease (vWD) in Indian patients was studied. Three hundred and sixty-five unrelated normal individuals and 100 vWD patients (type 1: 18; type 2: 21; type 3: 61) were analyzed for the two vWF intron 40 VNTR markers. Polymerase chain reaction of the two markers vWF1 and vWF2 was done using specific primers followed by electrophoresis on 10% polyacrylamide gel. VNTR analysis revealed the presence of VNTR9 and VNTR15 along with the eight alleles VNTR6 to VNTR14 in the vWF1 marker. Furthermore, apart from the six alleles, VNTR1 to VNTR6 of the vWF2 marker, two new alleles, VNTR7 and VNTR8, were also found. The heterozygosity rates were 75 and 74% for vWF1 and vWF2, respectively. Overall, the heterozygosity rate, i.e. when both vWF1 and vWF2 were considered in combination, was 81%. These data were successfully applied for the detection of carriers in 10 severe type 3 vWD families. The high heterozygosity of the two vWF intron 40 VNTR markers and the simplicity of the technique without much cost being involved suggest the practical feasibility of this technique in developing countries like India.
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Iron deficiency is a continuum beginning from lowering of tissue stores to the phase of exhausted tissue stores, interference with iron driven biochemical reactions in the body, microcytosis, hypochromia, increasing severity of anaemia with all its attendant consequences. Iron deficiency anaemia is a very well known concept but what is often not appreciated is the effect of broad canvas of iron deficiency on various tissues, organs and systems in our body in addition to iron deficiency anaemia leading to concept of "Iron deficiency disease". In this condition not only tissue delivery of oxygen is compromised but proliferation, growth, differentiation, myelinogenesis, immunofunction, energy metabolism, absorption and biotransformation are compromised leading to abnormal growth and behaviour, mental retardation, reduced cardiac performance and work efficiency, infection etc which ultimately leads to the concept that "iron deficiency not only breaks the machine but also wrecks the machinery."
BACKGROUND AND OBJECTIVES: Afibrinogenemia and hypofibrinogenemia are rare inherited coagulation disorders characterized by hemorrhagic manifestations of variable entity and by plasma fibrinogen deficiency. So far, 57 mutations have been associated with these disorders, and 18 of these are missense mutations. The aim of this study was to characterize the molecular mechanism underlying severe hypofibrinogenemia in a proband from India. DESIGN AND METHODS: The mutational screening was accomplished by DNA sequencing of the three fibrinogen genes. The mutant protein was expressed in COS-1 cells, and intracellular and secreted mutant fibrinogen was analyzed by means of pulse-chase experiments. RESULTS: A novel homozygous G-->A transition in exon 8 (nucleotide position 8017) was found in the proband's fibrinogen Bbeta-chain gene. The resulting G434D missense mutation (fibrinogen Mumbai) involves a highly conserved amino acid residue, located in the C-terminal globular D domain. In vitro expression experiments demonstrated intracellular retention of the mutant fibrinogen and marked reduction of its secretion. INTERPRETATION AND CONCLUSIONS: The G434D substitution causes severe hypofibrinogenemia by impairing fibrinogen secretion. Expression data confirm the importance of Bbeta-chain D domain folding in the intracellular processing of fibrinogen.
Hb-M is a very rare hemoglobinopathy in the Indian subcontinent. We report a family with Hb-M with lifelong cyanosis from the Ratnagiri district in western India. The propositus was a 11-year-old female child with a history of increasing cyanosis exacerbated by fever and weakness. Similar complaints were also noted in her mother and five maternal family members. There was no history of cardiac illness or exposure to drugs and chemicals. The methemoglobin level was 39.3% in the propositus and 21.1% in her mother with normal NADH-methemoglobin reductase activity. Abnormal absorption peaks by spectroscopic analysis, presence of hemoglobin instability, and a slow-moving band on starch gel electrophoresis supported the presence of Hb-M. Automated DNA sequence analysis of the beta globin gene showed a C-->T substitution at codon 63. This leads to a substitution of histidine (CAT) by tyrosine (TAT) at the beta 63 (E7) position, similar to Hb-M Saskatoon. We have named this variant as Hb-M(Ratnagiri).
Combined deficiency of factors V (FV) and factor VIII (FVIII) (F5F8D) is an autosomal recessive bleeding disorder caused by simultaneous moderate-to-mild decrease of both clotting proteins. Mutations in two components of the ER-Golgi intermediate compartment (ERGIC-53), i.e., lectin mannose binding protein (LMAN1) and multiple coagulation factor deficiency 2 (MCFD2), have been found to be responsible for this dual deficiency in most of the cases reported in literature. Three Indian families with F5F8D were analyzed for the presence of mutations in their LMAN1 and MCFD2 genes. One of the three families showed the presence of a G to A substitution in exon 2 of the MCFD2 gene, whereas another family showed a nonsense mutation, i.e., G to T substitution, in exon 2 of the LMAN1 gene, the latter being a novel mutation not previously reported. The third family did not show mutations in either of the two genes, suggesting that a significant subset of F5F8D cases may be due to additional genes resulting in a similar phenotype.
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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.
In order to evaluate the incidence of hereditary bleeding disorders in patients presenting with menorrhagia, where the usual gynecological and endocrinal causes of bleeding were ruled out by various local ultrasonography (USG) and relevant endocrine investigations, 120 women aged between 18 and 35 years presenting with menorrhagia without any discernable cause were studied using an open design, where the investigators knew that these patients had menorrhagia. These patients were investigated for inherited coagulation defects. Of the 120 women investigated, 19.16% (23 cases) had an inherited coagulation disorder to account for their menorrhagia. Although a majority (11.6%) are patients with von Willebrand's disease (VWD), other rare platelet disorders such as Glanzmann's thrombasthenia (3.3%), Bernard-Soulier syndrome (0.83%), coagulation factor deficiencies such as factor VIII (0.83%), factor X (0.83%), and factor XI (0.83%), and immune thrombocytopenia (0.83%) were also found. Taking a detailed history for bleeding from other sites however minor, paternal consanguinity as well as family history of bleeding tendencies appeared as a very strong predictor for such kinds of disease in patients with menorrhagia. Patients with menorrhagia without a discernable cause, therefore, need evaluation for the congenital coagulation disorders.
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The present survey, which involves 70 sickle cell anemia patients from Gujarat and Maharashtra revealed a high prevalence of the typical Arab-Indian haplotype (91.5 %). Six atypical haplotypes, including a Cameroon one were also found. Correlation of these various haplotypes with HbF expression was studied.