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Objective This study aimed to offer genetic diagnosis to affected type 3 severe von Willebrand disease families. Method Thirteen families were referred for prenatal diagnosis during the first and second trimesters of pregnancy. Prenatal diagnosis was offered by chorionic villus sampling between 11 and 12 weeks and by cordocentesis between 18 and 19.5 weeks of gestation. Phenotypic analysis included FVIII:C and von Willebrand factor antigen assays. A combination of molecular biological techniques which included PCR-restriction fragment length polymorphism technique using intron 40 variable number tandem repeat (VNTR) markers, conformation sensitive gel electrophoresis, direct DNA sequencing, and multiple ligation probe amplification (MLPA) were used to offer genotyic diagnosis in the remaining families. Results Diagnosis was offered by intron 40 VNTR analysis in eight families. In one family, the diagnosis was given by direct mutation detection technique, whereas in another diagnosis was given by MLPA technique as the index case showed the presence of large deletion within von Willebrand factor. In three families, diagnosis was offered by cordocentesis on the basis of phenotypic assays, further confirmed by genotyping. Conclusion Both first and second-trimester prenatal diagnoses could be successfully offered using a combination of phenotypic and genotypic techniques to all severe von Willebrand disease families. © 2014 John Wiley & Sons, Ltd.

Acquired von Willebrand disease (AVWD) is a rare, underdiagnosed hemorrhagic disorder, which is similar to congenital VWD with regard to the clinical and laboratory parameters; however, it is found in individuals with no positive family history and has no genetic basis. The etiology is varied, the commonest being hematoproliferative disorders and cardiovascular disorders. Other disorders associated with AVWD are autoimmune disorders such as systematic lupus erythematosus, hypothyroidism, and neoplasia, or it may also be drug induced. In quite a few cases, the etiology is unknown. The pathogenic mechanisms are different in different underlying disorders or they may be overlapping among these disorders. Some of the proposed mechanisms include the development of autoantibodies, selective absorption of high molecular weight von Willebrand factor (VWF) multimers, non-selective absorption of VWF, mechanical destruction of VWF under high shear stress, and increased proteolysis. This report presents a concise review of the pathophysiological mechanisms of AVWD in these various underlying conditions.

The JAK2 V617F mutation is recurrent in polycythemia vera and essential thrombocythemia, which are myeloproliferative neoplasms frequently associated with arterial and venous thromboembolism. It has also been reported as a marker for occult myeloproliferative disorder (MPD) in patients with splanchnic venous thrombosis. Limited data are available regarding the prevalence of the JAK2 V617F mutation in patients with thrombosis outside the splanchnic region. For the study, 321 cases of venous thrombosis in the splanchnic and nonsplanchnic regions (cerebral venous thrombosis [CVT], 70; deep venous thrombosis [DVT], 36; Budd-Chiari syndrome [BCS], 137; portal venous thrombosis [PVT], 78) were studied for the presence of JAK2 mutations. The prevalence values for the JAK2 mutation were 3% (1/36), 8.8% (12/137), 5% (4/78), and 3% (2/70) in DVT, BCS, PVT, and CVT, respectively; 19 (5.9%) of 321 cases were positive for the JAK2 mutation. Of 111 healthy subjects screened for this mutation, none were found to be carriers. Determination of the JAK2 V617F mutation may be useful to identify patients who should be carefully observed for the development

A serious complication of replacement therapy in patients with bleeding disorders is the development of ‘inhibitors’, particularly FVIII inhibitors in haemophilia A patients. This leads to an increase in the management cost, morbidity and mortality, especially post-operatively. The mechanism of FVIII inhibitor development is quite complex and it is difficult to predict inhibitor development, but a prompt and accurate diagnosis is critical as early therapy can save lives. The aim of this study was to screen patients with bleeding disorders in India for inhibitors, and to analyse and compare the prevalence of inhibitors in different regions in India. Patient details were recorded and blood samples were collected in sodium citrate vacutainers from 1,505 patients with bleeding disorders, in different cities in India. Coagulation and inhibitor screening assays were performed, followed by the Bethesda assay in inhibitor positive samples to quantify the FVIII inhibitor titre. Out of the 1,505 samples analysed, 1,285 were Haemophilia A patients, out of which 78 (6.07 %) were positive for ‘FVIII Inhibitors’. The highest incidence of FVIII Inhibitors was seen in South India (13.04 %). The highest incidence of 20.99 % was observed in Chennai, followed by Hyderabad (13.33 %), Jammu (9.90 %) and Guwahati (8.51 %), respectively, with respect to the samples analysed. The other regions showed an inhibitor incidence

Delayed haemorrhage due to vitamin K deficiency in early infancy has rarely been the cause of acquired hemostatic disorders. We report here 11 cases of vitamin K deficiency bleeding (VKDB), despite receiving appropriate dosage of injectible vitamin K at birth. Bleeding symptoms ranged from excessive bleed from cuts to intracranial bleed. Tuberculosis, diarrhea with intermittent antibiotic therapy were some of the associated symptoms. Laboratory tests confirmed acquired bleeding diathesis due to vitamin K deficiency, which was corrected after adequate vitamin K supplementation. VKDB is not an uncommon phenomenon and should be considered in the differential diagnosis of a child with bleeding diathesis.

Dear Editor, The human von Willebrand factor gene (VWF) is located on the short arm of chromosome 12 and spans approximately 178 kb, comprising of 52 exons. The mRNA has a length of 8.7 kb coding for precursor protein of 2813 amino acids that includes a 22 amino acid signal peptide, a pro-peptide of 741 amino acids and a mature subunit of 2050 amino acids [1]. The gene is highly polymorphic with more than 2000 polymorphisms reported in different ethnic populations [2]. Single nucleotide variations (SNVs) contribute to normal phenotypic variations and diseases as well. A chance combination of two SNVs in a single codon changing the nature of the mutation though uncommon, yet higher prevalence of the variant alleles across populations, makes such a deleterious combination possible resulting in the disease phenotype. We report here a severe VWD patient (VWF TGA) by the presence of two heterozygous changes (c.2878C>T, rs370984712; c.2880G>A, rs1800380) in the same codon. The single change c.2878C>T results in a variant (p.R960W), while c.2880G>A results in a synonymous change (p.R960=). The effect of SNV c.2878C>T on the function of VWF is unclear though it has been reported as a polymorphism in the single nucleotide polymorphism database (NCBI dbSNP. NM_000552.3); the prediction softwares however showed the effect of this change as deleterious. Only one such case has been reported earlier wherein the patient was found to have two mutations, i.e., a partial deletion extending the whole gene, i.e., exons 1–52 in heterozygous condition and R960* in homozygous condition, though the details of the second mutation are not discussed in this report [4]. The allelic frequencies of c.2880G>A are quite high varying between 0 and 32 % in different populations, while the frequency of c.2878C>T has been reported to be quite low, i.e., 0.05 % (http://asia.ensembl.org/Homo_sapiens/Info/ Index). One limitation here is that the parents were unavailable for mutation studies, but it is logical to presume that both the variants were in cis position, as the whole gene was sequenced and the patient was found to carry only two single nucleotide variations in heterozygous state. In conclusion, we report an interesting mechanism of two single nucleotide variations occurring together in cis in a single codon changing the nature of the pathogenic mutation. In the presence of the first variant, it would have been a missense variant, probably pathogenic, but the coexistence of a second SNV has changed the nature of the mutation, i.e., from a missense to a premature termination codon mutation. The two SNVs are mutually * Shrimati Shetty shrimatishetty@yahoo.com

Background Though von Willebrand disease (VWD) is a common coagulation disorder, due to the complexity of the molecular analysis of von Willebrand factor gene (VWF), not many reports are available from this country. Large size of the gene, heterogeneous nature of mutations and presence of a highly homologous pseudogene region are the major impediments in the genetic diagnosis of VWD. The study is aimed at unravelling the molecular pathology in a large series of VWD patients from India using an effective strategy. Method We evaluated 85 unrelated Indian type 3 VWD families to identify the molecular defects using a combination of techniques i.e. PCR-RFLP, direct DNA sequencing and multiple ligation probe amplification (MLPA). Results Mutations could be characterized in 77 unrelated index cases (ICs). 59 different mutations i.e. nonsense 20 (33.9%), missense 13 (22%), splice site 4 (6.8%), gene conversions 6 (10.2%), insertions 2 (3.4%), duplication 1 (1.7%), small deletions 10 (17%) and large deletions 3 (5.1%) were identified, of which 34 were novel. Two common mutations i.e. p.R1779* and p.L970del were identified in our population with founder effect. Development of alloantibodies to VWF was seen in two patients, one with nonsense mutation (p.R2434*) and the other had a large deletion spanning exons 16–52. Conclusion The molecular pathology of a large cohort of Indian VWD patients could be identified using a combination of techniques. A wide heterogeneity was observed in the nature of mutations in Indian VWD patients.

This study aimed to offer genetic diagnosis to affected type 3 severe von Willebrand disease families.Thirteen families were referred for prenatal diagnosis during the first and second trimesters of pregnancy. Prenatal diagnosis was offered by chorionic villus sampling between 11 and 12 weeks and by cordocentesis between 18 and 19.5 weeks of gestation. Phenotypic analysis included FVIII:C and von Willebrand factor antigen assays. A combination of molecular biological techniques which included PCR-restriction fragment length polymorphism technique using intron 40 variable number tandem repeat (VNTR) markers, conformation sensitive gel electrophoresis, direct DNA sequencing, and multiple ligation probe amplification (MLPA) were used to offer genotyic diagnosis in the remaining families.Diagnosis was offered by intron 40 VNTR analysis in eight families. In one family, the diagnosis was given by direct mutation detection technique, whereas in another diagnosis was given by MLPA technique as the index case showed the presence of large deletion within von Willebrand factor. In three families, diagnosis was offered by cordocentesis on the basis of phenotypic assays, further confirmed by genotyping.Both first and second-trimester prenatal diagnoses could be successfully offered using a combination of phenotypic and genotypic techniques to all severe von Willebrand disease families.

Acquired von Willebrand disease (AVWD) is a rare, underdiagnosed hemorrhagic disorder, which is similar to congenital VWD with regard to the clinical and laboratory parameters; however, it is found in individuals with no positive family history and has no genetic basis. The etiology is varied, the commonest being hematoproliferative disorders and cardiovascular disorders. Other disorders associated with AVWD are autoimmune disorders such as systematic lupus erythematosus, hypothyroidism, and neoplasia, or it may also be drug induced. In quite a few cases, the etiology is unknown. The pathogenic mechanisms are different in different underlying disorders or they may be overlapping among these disorders. Some of the proposed mechanisms include the development of autoantibodies, selective absorption of high molecular weight von Willebrand factor (VWF) multimers, non-selective absorption of VWF, mechanical destruction of VWF under high shear stress, and increased proteolysis. This report presents a concise review of the pathophysiological mechanisms of AVWD in these various underlying conditions.

The JAK2(V617F)mutation is recurrent in polycythemia vera and essential thrombocythemia, which are myeloproliferative neoplasms frequently associated with arterial and venous thromboembolism. It has also been reported as a marker for occult myeloproliferative disorder (MPD) in patients with splanchnic venous thrombosis. Limited data are available regarding the prevalence of the JAK2(V617F) mutation in patients with thrombosis outside the splanchnic region. For the study, 321 cases of venous thrombosis in the splanchnic and nonsplanchnic regions (cerebral venous thrombosis [CVT], 70; deep venous thrombosis [DVT], 36; Budd-Chiari syndrome [BCS], 137; portal venous thrombosis [PVT], 78) were studied for the presence of JAK2 mutations. The prevalence values for the JAK2 mutation were 3% (1/36), 8.8% (12/137), 5% (4/78), and 3% (2/70) in DVT, BCS, PVT, and CVT, respectively; 19 (5.9%) of 321 cases were positive for the JAK2 mutation. Of 111 healthy subjects screened for this mutation, none were found to be carriers. Determination of the JAK2(V617F) mutation may be useful to identify patients who should be carefully observed for the development of overt MPDs. The significance of screening for this mutation in nonsplanchnic thrombosis cases needs to be analyzed in a larger series.