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M Chandy

Publications and source records attributed to M Chandy.

At least 19 recordsLinked to original sources
PubMed2006

Developing an algorithm of informative markers for evaluation of chimerism after allogeneic bone marrow transplantation.

Analysis of chimerism by polymerase chain reaction amplification of STR or VNTR has become a routine procedure for the evaluation of engraftment after allogeneic stem cell transplantation. Knowledge of the frequency of different STR or VNTR alleles in unrelated individuals in a population is useful for forensic work. In the context of HLA identical sibling bone marrow transplantation the informativeness of these markers needs to be evaluated. We evaluated five STRs (THO1, VWA, FES, ACTBP2, and F13A1) and 1 VNTR (APOB) for informativeness in stem cell transplants from HLA identical sibling donors. All four markers used individually allowed us to discriminate 20-56% of the patient donor pairs. Using a combination of all these markers along with a polymorphic marker in the beta-globin gene and the sex chromosome specific amelogenin marker, we were able to discriminate 99% of the patient donor pairs. We have established an algorithm for evaluating chimerism following HLA identical sibling donor transplants in the Indian population using molecular markers in 310 patients. Analysis of heterozygote frequencies in different populations is similar suggesting that this algorithm can be used universally for transplant centers to evaluate chimerism following allogeneic bone marrow transplantation.

Algorithms↗
PubMed2006

An approach to the management of leukemia in the developing world.

Management of leukemia is expensive. In the developing world where there are significant resource constraints, how does the physician manage leukemia? Government health budgets need to put their money into areas where a small investment is likely to benefit a large proportion of its people and these would include immunization, sanitation, family planning and treatment of communicable diseases and the cost of expensive treatments would have to be borne by the patient and family. However, in many of the emerging economies in the developing world, society is three-tiered in terms of resources with a small proportion being able to afford the best treatment, the middle class with the ability to meet a significant proportion of the healthcare costs on their own and the poor who depend wholly on what the Government health budget can afford. Providing an accurate diagnosis is possible even with limited resources and this must be made available to all. Sometimes a precise diagnosis even at the molecular level is more important in the developing world so that resources are not wasted on inappropriate treatments. Choosing a protocol for management based on resources available for the individual patient would appear ethically unacceptable, but is probably the only solution when wealth is unevenly distributed in society. This paper illustrates an approach to the management of leukemia in the developing world that has evolved with experience over two decades in a tertiary referral hospital in India.

Antineoplastic Protocols↗
PubMed2006

Genetic diagnosis of haemophilia and other inherited bleeding disorders.

Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency. Haemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII) and factor IX (FIX). Together with von Willebrand's disease, a defect of primary haemostasis, these X-linked disorders include 95% to 97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits, are rare with prevalence of the presumably homozygous forms in the general population of 1:500,000 for FVII deficiency and 1 in 2 million for prothrombin (FII) and factor XIII (FXIII) deficiency. Molecular characterization, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children affected by coagulation disorders in developing countries, where patients with these deficiencies rarely live beyond childhood and where management is still largely inadequate. These characterizations are possible by direct or indirect genetic analysis of genes involved in these diseases, and the choice of the strategy depends on the effective available budget and facilities to achieve a large benefit. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for mutation detection. However, in countries with limited facilities and low budget resources, carrier detection and prenatal diagnosis are usually performed by linkage analysis with genetic markers. This article reviews the genetic diagnosis of haemophilia, genetics and inhibitor development, genetics of von Willebrand's disease and of rare bleeding disorders.

Blood Coagulation Disorders, Inherited↗
PubMed2006

Diversity of Glanzmann thrombasthenia in southern India: 10 novel mutations identified among 15 unrelated patients.

BACKGROUND: Glanzmann thrombasthenia (GT) is a congenital bleeding disorder caused by either a lack or dysfunction of the platelet integrin alphaIIbbeta3. OBJECTIVES: To determine the molecular basis of GT in patients from southern India. PATIENTS: Fifteen unrelated patients whose diagnosis was consistent with GT were evaluated. RESULTS: Platelet surface expression of alphaIIbbeta3 was < 10%, 10%-50%, and > 50% of controls in five, nine, and one patient(s), respectively. Immunoblotting of the platelet lysates showed no alphaIIb in 14 patients, and no beta3 in 10 patients, although severely reduced in four patients. Platelet fibrinogen was undetectable in 13 patients, and severely reduced in one patient. One patient showed normal surface alphaIIbbeta3 expression, and normal alphaIIb, beta3 and fibrinogen levels in the lysate. Ten novel candidate disease-causing mutations were identified in 11 patients. The missense mutations included Gly128Ser, Ser287Leu, Gly357Ser, Arg520Trp, Leu799Arg in alphaIIb, and Cys575Gly in beta3. We have already shown that Gly128Ser, Ser287Leu, and Gly357Ser mutations variably affect alphaIIbbeta3 surface expression. The Cys575Gly mutation may disrupt the disulphide link with Cys586 to cause the GT phenotype. The molecular pathology of the other missense mutations is not clear. Two nonsense mutations, Trp-16Stop and Glu715Stop in alphaIIb, and a 7-bp deletion (330-336TCCCCAG) in beta3 are predicted to result in truncated proteins. An IVS15(-1)G --> A mutation in alphaIIb induced a cryptic splice site as confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Thirteen polymorphisms were also identified (five in alphaIIb and eight in beta3), among which five were novel. CONCLUSIONS: While identifying a significant number of novel mutations causing GT, this study confirms the genetic heterogeneity of the disorder in southern India.

Adolescent↗
PubMed2006

Molecular screening of the neutrophil elastase gene in congenital neutropenia.

Congenital neutropenia is a rare hematopoietic disease, which occurs sporadically or as an auto-somal dominant inherited disorder. Pathogenesis of congenital neutropenia can now be attributed to mutations of the ELA2 gene encoding neutrophil elastase. A child with severe congenital neutropenia with a heterozygous mutation G1887A in exon 2 of ELA2 gene is reported.

DNA Mutational Analysis↗
PubMed2005

Six novel mutations including triple heterozygosity for Phe31Ser, 514delT and 516T-->G factor X gene mutations are responsible for congenital factor X deficiency in patients of Nepali and Indian origin.

Factor X (FX) deficiency is a rare (1 : 100000) autosomal recessive disorder caused by heterogeneous mutations in FX gene. We have studied the molecular basis this disease in six Indian and one Nepali patients. Diagnosis was confirmed by measuring the FX coagulant activity (FX: C) using a PT based assay. Six of them had a FX: C of < 1% and one patient had 24% coagulant activity. Mutations were identified in all the seven patients. These included eight (88.8%) missense and one frame-shift (11.2%) mutations of which six were novel. Three of the novel mutations, a Phe31Ser affecting 'Gla' domain and 514delT and 516T-->G mutations affecting Cys132 in 'connecting region' were identified in a triple compound heterozygous state in a Nepali patient presenting with a severe phenotype. Two other novel mutations, Gly133Arg, may affect the disulphide bridge between Cys132-Cys302 in the connecting region while Gly223Arg may perturb the catalytic triad (His236, Asp282 and Ser379). The other novel mutation, Ser354Arg, involves the replacement of a small-buried residue by a large basic aminoacid and is likely to have steric or electrostatic effects in the pocket involving Lys351-Arg347-Lys414 that contributes to the core epitope of FXa for binding to FVa. Three previously reported mutations, Thr318Met; Gly323Ser; Gly366Ser were also identified. This is the first report of the molecular basis of FX deficiency in patients from the Indian subcontinent.

Arginine↗
PubMed2005

Fludarabine-based conditioning for allogeneic stem cell transplantation for multiply transfused patients with Fanconi's anemia.

A fludarabine-based protocol (fludarabine (25 mg/m(2)/day x 6 days), cyclophosphamide (10 mg/kg/day x 2 days) and ATG (ATGAM 10 mg/kg/day x 4 days)) was used in four multiply transfused Fanconi's anemia (FA) patients aged 5-15 years to reduce rejection during allogeneic bone marrow transplantation (BMT). Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and mini methotrexate. The graft source was G-CSF-stimulated bone marrow or peripheral blood stem cells (PBSC) in two patients each. All patients engrafted with median time to ANC>500/mm(3) being 14 days (range: 12-17) and unsupported platelet count >20 ,000/mm(3) being 13 days (range: 11-18). One patient had secondary graft rejection on day 56 and expired on day 69 due to fungal pneumonia. One patient who developed acute myeloid leukemia on day 56 underwent successful induction with cytosine and daunorubicin followed by peripheral blood stem cell (PBSC) rescue on day 70 and is presently in remission with complete donor chimerism and grade I GVHD. At a median follow-up of 13 months (range: 4-21), three patients (75%) are well with complete donor chimerism. Addition of fludarabine to the conditioning regimen for BMT in FA can provide additional immunosuppression for engraftment without increasing toxicity.

Adolescent↗
PubMed2005

Pre-transplant reduction of isohaemagglutinin titres by donor group plasma infusion does not reduce the incidence of pure red cell aplasia in major ABO-mismatched transplants.

Major ABO incompatibility in stem cell transplant recipients has been associated with pure red cell aplasia (PRCA). Reduction of incompatible isohaemagglutinin titres pre-transplant by various methods has been thought to reduce the incidence of PRCA. Our data suggest that pre-transplant reduction of incompatible isohaemagglutinin titres by donor group plasma infusion does not reduce the incidence of PRCA. We also failed to find any relationship between pre-transplant ABO isohaemagglutinin titre and the risk of developing PRCA.

ABO Blood-Group System↗
PubMed2005

Randomized trial of two different conditioning regimens for bone marrow transplantation in thalassemia--the role of busulfan pharmacokinetics in determining outcome.

In total, 94 patients with homozygous beta thalassemia were randomized to two different conditioning regimens: busulfan 600 mg/m2 + cyclophosphamide 200 mg/kg or busulfan 16 mg/kg + cyclophosphamide 200 mg/kg and antilymphocyte globulin (47 in each group), for bone marrow transplantation, to see whether increased myeloablation or increased immunosuppression would reduce rejection. Busulfan pharmacokinetics in determining outcome was evaluated. There was no significant difference in engraftment, graft-versus-host disease, rejection, and overall and disease-free survival in the two groups. Systemic exposure to busulfan was significantly higher in the 600 mg/m2 group, but in both groups there was a wide interindividual variation in the busulfan kinetics. Six patients rejected the graft, two in the busulfan 600 mg group and four in busulfan 16 mg group (P = 0.677 CI -0.17, 0.07), but in five patients (pharmacokinetic data not available in one patient) who rejected the graft busulfan first dose trough level (C(min)-1) was below 150 ng/ml while it was above this level in the 66 of 68 patients with successful engraftment (P < or = 0.001). This randomized trial shows that rejection is influenced by busulfan levels and suggests that monitoring of busulfan levels and dose adjustment based on first-dose kinetics may reduce the risk of rejection.

Antilymphocyte Serum↗
PubMed2005

Identification of factor VIII gene mutations in 101 patients with haemophilia A: mutation analysis by inversion screening and multiplex PCR and CSGE and molecular modelling of 10 novel missense substitutions.

Haemophilia A (HA) is an X-linked bleeding disorder caused by diverse mutations in the human coagulation factor VIII (FVIII) gene. We have analysed DNA from 109 unrelated Indian patients with HA for their FVIII gene defects. Among these patients 89 (82%) had severe (FVIII:C <1%) HA, 11 (10%) had moderate (FVIII:C 1-5%) HA and nine (8%) had mild (FVIII:C 5-30%) HA. These patients were first screened for the common intron 22 and intron 1 inversions. Inversion negative samples were screened for point mutations by a multiplex PCR and conformation sensitive gel electrophoresis strategy. Mutations were identified in 101 of the 109 patients. These included two (2%) intron 1 and 51 (51%) intron 22 inversions, four (4%) gross deletions and 44 (43%) point mutations. Twenty-nine novel causative mutations, including 11 missense, seven frameshift, five nonsense mutations, three splice site defects and three gross deletions were detected. Ten of the novel missense mutations were studied by molecular modelling. Two different (Thr2253Pro and Pro1392fs) mutations were seen in four unrelated families and FVIII gene haplotyping suggested a common founder effect. Seven of these 109 patients had inhibitors. Among them, four had intron 22 inversions, one had a novel gross deletion (delexon 2-9) and one a nonsense mutation (Trp1535Stop). In one of these patients, no mutation could be identified in the FVIII gene. A Thr2253Pro novel mutation and an intron 22 inversion were identified in two female haemophiliacs. The data from this study suggests that the spectrum of gene defects in Indian patients with HA is as heterogeneous as reported in other populations.

Amino Acid Substitution↗
PubMed2005

Molecular genetics of hereditary prothrombin deficiency in Indian patients: identification of a novel Ala362 --> Thr (Prothrombin Vellore 1) mutation.

Prothrombin deficiency is a rare (1:200 000) autosomal recessive disorder caused by diverse mutations in prothrombin gene. We have studied the molecular basis of this disorder in four unrelated Indian patients. The diagnosis was based on prolonged prothrombin (PT) and activated partial thromboplastin times and low factor II coagulant activity (FII: C) measured using a PT based assay. FII: C levels ranged between 4.7% and 17.5%. Mutations were identified in all the four patients. Five different causative mutations including four (80%) missense and an in-frame deletion (20%) were identified. One of them was a novel, Ala362 --> Thr amino acid change affecting 'B' chain of -thrombin. This mutation was present in a compound heterozygous state with a previously reported Arg-1 --> Gln missense change affecting pro-peptide cleavage site. Ala362 --> Thr occurred at a codon, evolutionarily conserved in all the 24 different prothrombins or its related serine proteases studied. Molecular modeling of this mutation was found to cause a conformational change around the region involving a catalytic triad residue His363 and a cysteine residue at codon 364. The FII: C level in this patient was 17.5%. Three other previously reported mutations were also detected in the homozygous state: Arg271 --> Cys in Kringle-2 region, a Glu309 --> Lys in "A" chain of -thrombin and an in-frame deletion of 3 bp (AAG) leading to Del Lys301/302 in "A" chain of -thrombin. This is the first report of the molecular basis of prothrombin deficiency in Indian patients and we suggest the eponym 'Prothrombin Vellore 1' for Ala362 --> Thr mutation.

Adult↗
PubMed2005

Three novel beta-propeller mutations causing Glanzmann thrombasthenia result in production of normally stable pro-alphaIIb, but variably impaired progression of pro-alphaIIbbeta3 from endoplasmic reticulum to Golgi.

BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder characterized by lack of platelet aggregation in response to most physiological agonists and caused by either a lack or dysfunction of the platelet integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa). OBJECTIVES: To determine the molecular basis of GT and characterize the mutations by in vitro expression studies. PATIENTS: We studied three unrelated patients from southern India whose diagnosis was consistent with GT. RESULTS: Immunoprecipitation of the cell lysates and immunoblotting showed no detectable mature alpha(IIb) in the G128S mutant, in contrast to 6% and 33% of the normal amount of mature alpha(IIb) in the S287L and G357S mutants, respectively. Pulse-chase analysis demonstrated pro-alpha(IIb) in the mutants comparable with the normal pro-alpha(IIb), but no conversion to mature alpha(IIb) in the G128S mutant, and only trace conversion to mature alpha(IIb) in the S287L and G357S mutants. The disappearance of pro-alpha(IIb) in the three mutants was similar to that in cells expressing normal alpha(IIb)beta3 or alpha(IIb) only. All three mutants demonstrated pro-alpha(IIb)beta3 complexes and co-localized with an ER marker by immunofluorescence. The G128S mutant showed no co-localization with a Golgi marker, and the other two mutants showed minimal and moderate co-localization with the Golgi marker. CONCLUSIONS: These three beta-propeller mutations do not affect the production of pro-alpha(IIb), its ability to complex with beta3, or its stability, but do cause variable defects in transport of pro-alpha(IIb)beta3 complexes from the endoplasmic reticulum to the Golgi.

Adult↗
PubMed2005

Cytochrome P4501A1 and glutathione S transferase gene polymorphisms in patients with aplastic anemia in India.

The etiology of acquired aplastic anemia (AA) in most patients remains unclear. It is believed that patients with a reduced ability to detoxify environmental toxins are at increased risk of developing AA. Cytochrome P450 (CYP450) and glutathione S transferase (GST) are the major phase I and phase II xenobiotic-metabolizing enzymes. We analyzed the impact of the polymorphisms in CYP4501A1 and GSTM1 and GSTT1 genes on the susceptibility and disease severity in 200 patients with AA and compared the frequency with the normal population. There was a significantly increased frequency of the CYP1A1m4 allele in AA patients compared with normal controls (odds ratio = 3.01; 95% confidence interval 1.76-5.17; p = 0.00001). None of the other CYP1A1 genotypes or the GST genotypes were significantly different between AA patients and controls. Altered metabolism of benzo(a)pyrene due to the polymorphism in the CYP1A1 gene might be an etiologic factor in the increased incidence of AA in these patients. The CYP1A1m4 allele may play a role in determining the risk of AA in India.

Adolescent↗