Type-3 von Willebrand disease in India-Clinical spectrum and molecular profile.

INTRODUCTION: Type 3 von Willebrand disease (VWD) is the rare and most severe form of VWD which results from a near-complete deficiency of the von Willebrand factor (VWF). This study evaluates in detail the molecular pathology of type-3 VWD in India. One hundred and two patients from 90 families were evaluated. PATIENTS AND METHODS: Phenotypic data, including bleeding scores (BS), were documented using structured questionnaires. Diagnosis of type 3 VWD was based on undetectable VWF antigen levels in the plasma. Genomic DNA from these patients was screened for mutations in VWF gene. Structural modeling and expression studies were carried out for missense mutations. RESULTS: Out of 102 patients, mutations could be identified in 91% (n = 93). Fifty-five different gene variants were identified. Thirty-four (61.8%) were novel. Mutations could be identified in both the alleles in 90 patients, while no causative mutation could be identified in 9 patients; twenty-four (23.5%) patients had mutations clustered in the propeptide region of VWF. Interestingly, five mutations accounted for the defects in 37/93 (39.8%) patients. Structural analysis and in vitro studies on missense mutations imply impaired processes associated with secretion of VWF. CONCLUSION: This study is one of the largest series to define the molecular basis of type-3 VWD.

Molecular basis of quantitative fibrinogen disorders in 27 patients from India.

Congenital fibrinogen deficiency is an extremely rare (1:1 000 000) hereditary bleeding disorder caused by defects in genes coding for fibrinogen Aα-, Bß- and γ-chains, respectively. We report here the molecular basis of fibrinogen deficiency in a large series of patients from India. Twenty-seven patients with clinical features suggestive of fibrinogen deficiency and with prolonged plasma clotting times and low fibrinogen levels were studied. Genomic DNA was screened for mutations in the fibrinogen alpha (FGA), beta (FGB), gamma (FGG) genes by PCR and conformation sensitive gel electrophoresis. Fourteen different disease-causing mutations including frameshifts (51.9%), splice site (22.2%), missense (18.5%) and nonsense mutation (7.4%) were identified in 27 patients. Thirteen of them were novel, including seven frameshifts (fibrinogen Aα: p.Asp296 fs*59, p.Thr466 fs*17 and p.Lys575 fs*74; fibrinogen Bß: p.Gly414 fs*2 and fibrinogen γ: p.Ser81 fs*5, p.Lys185 fs*13 and p.Asp278_279 fs*17), three splice site mutations (FGA gene c.364+1G>A; c.510+2 T>G; FGB gene c.851+1G>A), two missense substitutions (fibrinogen Bß: p.Gly288Ser; p.Arg445Thr) and a nonsense mutation in fibrinogen Aα (p.Tyr127*). Two common mutations (FGA: c.364+1G>A, n = 6, FGG: p.Lys185 fs*13, n = 7) affecting 13 patients were identified in this series, suggesting that these mutations could be screened first in Indian patients with fibrinogen deficiency. The molecular data presented here is the largest series of patients with fibrinogen deficiency reported so far, adding significantly to the mutation database of this condition. It also helps create an algorithm for its genetic diagnosis in India.

Correlating clinical manifestations with factor levels in rare bleeding disorders: a report from Southern India.

Data on the clinical manifestations of patients with clotting factor defects other than Haemophilia A, B and von Willebrand disease are limited because of their rarity. Due to their autosomal recessive nature of inheritance, these diseases are more common in areas where there is higher prevalence of consanguinity. There is no previous large series reported from southern India where consanguinity is common. Our aim was to analyze clinical manifestations of patients with rare bleeding disorders and correlate their bleeding symptoms with corresponding factor level. Data were collected in a standardized format from our centre over three decades on 281 patients who were diagnosed with rare bleeding disorders (fibrinogen, prothrombin, factor V (FV), FVII, FX, FXI, FXIII and combined FV or FVIII deficiency). Patients with liver dysfunction or those on medications which can affect factor level were excluded. All patients with <50% factor levels were included in this analysis. Patients were analysed for their salient clinical manifestations and it was correlated with their factor levels. The data shows that FXIII deficiency is the commonest and FXI deficiency is the rarest in Southern India. There was no significant difference in bleeding symptoms among those who were < or >1% factor coagulant activities among all disorders, except for few symptoms in FVII and FX deficiency. An international collaborative study is essential to find out the best way of classifying severity in patients with rare bleeding disorders.

A patient-prioritized ability assessment in haemophilia: the Canadian Occupational Performance Measure.

Assessment of musculoskeletal function in individuals with haemophilia has been attempted with clinimetric instruments, which use predetermined domains for assessing the same. This study introduces the application of an instrument, the Canadian Occupational Performance Measure (COPM), which is an open-ended questionnaire that allows patients to prioritize their needs and rate their performance in different tasks of daily living as well as their satisfaction in performing them. To study the utility of COPM in evaluating the musculoskeletal functional status of patients with haemophilia and to assess its effectiveness in planning individualized management plans for them. COPM was administered to 67 individuals with haemophilia aged 10-55 years and the data were compared with functional deficits identified through FISH (Functional Independence Score for Haemophilia). A total of 31 performance difficulties in the areas of self-care (62%), productivity (21%) and leisure (17%) were identified by COPM. All eight domains of FISH were identified in COPM as problems in self-care. In addition to these, COPM identified problems in the areas of productivity and leisure. In 78% of the responses on COPM, there was concordance between the performance and satisfaction scores. However, there was discordance between the two in the remaining 22% of responses. COPM is a useful tool for assessment of musculoskeletal dysfunction in haemophilia. It provides a greater insight into the needs of each patient and helps in planning individualized intervention strategies.

Polymorphism in factor VII gene modifies phenotype of severe haemophilia.

The basis for 10-15% of patients with severe haemophilia having clinically mild disease is not fully understood. We hypothesized that polymorphisms in various coagulant factors may affect frequency of bleeding while functionally significant polymorphisms in inflammatory and immunoregulatory genes may also contribute to variations in the extent of joint damage. These variables were studied in patients with severe haemophilia, who were categorized as 'mild' (<5 bleeds in the preceding year, <10 World Federation of Haemophilia clinical and <10 Pettersson scores, n = 14) or 'severe' (all others, n = 100). A total of 53 parameters were studied in each individual for their association with the clinical severity. Age, F8:c activity and the incidence of thrombotic markers were comparable between the groups while the median number of bleeds, number of affected joints, clinical, radiological and functional joint scores (P < or = 0.001) and life-time clotting factor use (P < or = 0.007) were different. Patients with severe molecular defects had a 4.1-fold increased risk for a severe phenotype (95% CI: 1.18-14.42, P = 0.026) compared with other mutations. Of the polymorphisms studied, the FVII353Q (RR = 3.5, 95% CI: 1.04-12.05, P = 0.044) allele was associated with a severe phenotype. This data shows that apart from the F8/F9 genotype, functional polymorphisms in FVII gene affect the phenotype of patients with severe haemophilia.

Successful surgical haemostasis in patients with von Willebrand disease with Koate DVI.

This report describes our experience with Koate DVI, a factor VIII (FVIII) concentrate containing von Willebrand factor (VWF) for surgery in patients with von Willebrand's disease (VWD). Twenty-one patients underwent 26 procedures, 10 of which were major and 16 were minor. The median age was 27 years (3-55) and the mean weight was 52 kg (16-88). Among the ten patients (type 2-5; type 3-5) who underwent major procedures, the pre-operative dose was 35 IU kg(-1) of FVIII followed by 10-20 IU kg(-1) once daily depending on FVIII:C levels. The mean total dose of FVIII used per procedures was 106 IU kg(-1) (30-190) over a mean duration of 7 days (3-11). In this group, pre-infusion FVIII:C, VWF:Ag and VWF: ristocetin cofactor (RCoF) level that were 19.5% (1-64), 20 U dL(-1) (0-96) and 12% (0-66) increased to 72% (54-198), 131 U dL(-1) (68-206) and 68% (27-108) postinfusion, respectively. Sixteen minor procedures were performed in 11 patients (type 1-3, type 2-6, type 3-2). The preparative dose of FVIII was 10-20 IU kg(-1). The average duration of factor support was 2 days (1-3) for a mean total dose of 23 IU kg(-1) (9-60). The pre-infusion levels of FVIII:C, VWF:Ag and VWF:ristocetin cofactor (RCo) which were 31% (22-64), 25.5 U dL(-1) (0-63) and 21% (0-76), respectively, increased to 76% (27-111), 73 U dL(-1) (30-137) and 45% (2-106) postinfusion. Whereas surgical haemostasis was achieved in all patients, minor postoperative bleeding occurred after one procedure in each group. Both were controlled with additional doses of factor replacement. We conclude that Koate DVI in modest doses provide adequate haemostasis for surgery in patients with VWD.

Correction: Population pharmacokinetics of fludarabine in patients with aplastic anemia and Fanconi anemia undergoing allogeneic hematopoietic stem cell transplantation.

This article was originally published under a CC BY-NC-ND 4.0 license, but has now been made available under a CC BY 4.0 license. The PDF and HTML versions of the article have been modified accordingly.

Long-term outcome of mixed chimerism after stem cell transplantation for thalassemia major conditioned with busulfan and cyclophosphamide.

Mixed chimerism (MC) occurs frequently after allogeneic hematopoietic stem cell transplantation (HSCT) for thalassemia major (TM) and may be associated with rejection. We report the outcome of MC in 132 TM patients conditioned with Busulphan/Cyclophosphamide, who had successful engraftment and had ⩾1 year follow-up. Chimerism was first assessed at day +28, then every 3-9 months or more frequently if there was MC. If rejection was suspected, immunosuppression was stopped and donor-lymphocyte infusion (DLI) was given if there was no response. Among 132 patients, aged 7 years (range: 2-24), 46/132 (34.8%) had MC in the first year, 32/46 (69.6%) at day +28 and another 14 (30%) between day +28 and 1 year post HSCT. MC was quantified at level 1 (residual host chimerism (RHC) <10%) in 20 (43.5%), level II (RHC 10-25%) in 14 (30.4%) and level III (RHC >25%) in 12 (26.1%). On tapering immunosuppression, 15 (32.6%) developed acute GvHD and 8 (17.4%) had chronic GvHD with reversal to complete chimerism (CC). DLI was administered to 5/46 (10.9%), 1 evolved to CC but 4 rejected the graft. At median follow-up of 60 months (range: 16-172), 20/46 (43.5%) had CC, 18/46 (39.1%) had persistent MC with hemoglobin of 11.5 g/dL (range: 8.4-13.6), whereas 8 (17.4%) rejected the graft. Close monitoring and early intervention is needed with increasing recipient chimerism. Novel strategies are required for preventing graft rejection.

Hemostatic Abnormalities in Severe Renal Failure: Do They Bark or Bite?

Abnormal primary hemostasis is believed to be the most significant contributor to uremic bleeding. This study aimed to describe the prevalence and profile of primary and secondary hemostatic disorders in patients with chronic kidney disease (CKD) Stages 4 and 5 and to determine their association if any, with degree of uremia. Stages 4 and 5 predialysis CKD patients attending nephrology outpatient clinic were prospectively recruited and the following bleeding parameters were measured in all patients: platelet count, bleeding time (BT), Factor VIII assay, von Willebrand factor antigen (vWF:Ag), vWF:ristocetin cofactor activity (vWF:RCo), ratio of vWF:ristocetin cofactor activity to vWF antigen (vWF:RCo/vWF:Ag), prothrombin time (PT), and activated partial thromboplastin time (aPTT). Forty-five patients (80%, males) with a mean age of 39.4 years, 82% (n = 37) in Stage 5 CKD, were recruited for the study. The prevalence of thrombocytopenia was significantly higher among patients from West Bengal (15/26, 57.7%) compared to other study patients (2/19, 10.5%; P = 0.001); however, all had macrothrombocytes with normal BT, suggestive of the Harris syndrome. Factor VIII, vWF:Ag, vWF:RCo, vWF:RCo/vWF:Ag ratio, BT, PT, and aPTT were abnormal in 0 (0%), 0 (0%), 0 (0%), 4 (8.8%), 1 (2.2%), 7 (15.6%), and 5 (11.1%) patients, respectively. Except for thrombocytopenia, the prevalence of hemostatic abnormalities did not differ between CKD Stages 4 and 5. Hemostatic abnormalities are uncommon in Stages 4-5 CKD and except for thrombocytopenia, are not associated with degree of uremia. Constitutional macrothrombocytopenia is associated with normal BT even in CKD.

Fludarabine and cyclophosphamide based reduced intensity conditioning (RIC) regimens reduce rejection and improve outcome in Indian patients undergoing allogeneic stem cell transplantation for severe aplastic anemia.

Thirty-five patients (25 men and 10 women) with a median age of 20 years with severe aplastic anaemia (SAA) underwent HLA identical stem cell transplantation (HSCT) using a combination of fludarabine and cyclophosphamide +/- anti-thymocyte globulin between 2004 and 2006. Cyclosporine and mini methotrexate were used as GVHD prophylaxis. Graft source included peripheral blood stem cells (28) or G-CSF stimulated bone marrow (7). Two patients expired < 7 days post-HSCT while 32 (91.5%) patients engrafted with a median neutrophil and platelet engraftment time of 12 days each. Three patients (8.5%) developed veno-occlusive disease while acute GVHD occurred in 29% of evaluable patients, with chronic GVHD in 32%. At a mean follow-up of 22 months, 29 (82.8%) are alive and well. When compared with 26 patients previously transplanted using Cy200/antilymphocyte globulin, there was faster neutrophil engraftment (12 vs 16 days; P = 0.002) with significantly lower rejection rates (2.9 vs 30.7%; P = 0.003) and a superior event-free (82.8 vs 38.4%; P = 0.001) and overall survival (82.8 vs 46.1%; P = 0.005). A combination of fludarabine with cyclophosphamide +/- anti-thymocyte globulin reduces rejection and improves overall and event-free survival in Indian patients undergoing HSCT for severe aplastic anaemia.

Primary splenic marginal zone lymphoma with florid granulomatous reaction--a case report and review of literature.

Splenic marginal zone lymphomas (SMZL) constitute about 20% of primary splenic NHLs. We report a case of primary SMZL with a florid granulomatous reaction which obscured the underlying lymphoma. Although granulomas have been described in splenic non-Hodgkin lymphoma, it can be extensive and mask the underlying lymphoma. A careful search for the cytoarchitectural features of SMZL is warranted in such a case.

Population pharmacokinetics of fludarabine in patients with aplastic anemia and Fanconi anemia undergoing allogeneic hematopoietic stem cell transplantation.

Although hematopoietic stem cell transplantation (HSCT) with a conditioning regimen consisting of fludarabine (F-araA) and cyclophosphamide (Cy) is associated with improved outcome in young patients with aplastic anemia (AA) and Fanconi anemia (FA), several factors limit the success of the procedure. We evaluated the population pharmacokinetics (POPPK) of F-araA and its influence on HSCT outcome in patients (n=53) with AA and FA undergoing HSCT. Patients carrying a 5'-UTR polymorphism in NT5E gene (rs2295890 G>C) exhibited significantly lower plasma F-araA clearance compared to those with wild-type genotype (7.12 vs 5.03 L/h/m2 (29%) P<0.05). F-araA clearance was significantly higher in patients with AA compared to FA (2.46 ×, P<1e-6). Of all the outcome parameters evaluated (engraftment, rejection/graft failure, GvHD, TRM, OS), high F-araA AUC (>29.4 µm*h) was the only significant factor associated with the development of aGvHD by both univariate and multivariate analysis (P=0.02). The influence of plasma F-araA levels need to be evaluated in a larger cohort of patients to propose the need for therapeutic drug monitoring.

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.

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.

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.