We have studied a family with homozygous lethal, blood coagulation factor VII (FVII) deficiency. To identify the mutation responsible for the deficiency, exons 2 to 8 and the intron-exon junctions of their FVII genes were amplified from peripheral white blood cell DNA by polymerase chain reaction and screened by single-strand conformational polymorphism analysis. The fragment showing aberrant mobility was cloned and sequenced. We detected a single point mutation, a homozygous G to A substitution at nucleotide position 6070, in the invariant GT dinucleotide at the 5′ splice site of intron 4. Homozygosity was confirmed by loss of a site for the restriction endonuclease Mlu I. Analysis of the splicing pattern of ectopic transcripts in lymphocytes in the parents revealed that this mutation is associated with skipping of exon 4, which produces an mRNA encoding FVII with an in-frame deletion of the first epidermal growth factor–like domain (EGF 1). Transient transfection of COS-7 cells with an expression vector containing the ▵EGF 1 FVII cDNA shows that this mutant protein is not expressed. The identification of the molecular basis of the FVII deficiency in this family allowed mutation-specific prenatal diagnosis to be performed in a subsequent pregnancy. In this family complete FVII deficiency is associated with a severe bleeding diathesis but no developmental abnormalities, lending weight to the hypothesis that fetal FVII is not required for the putative angiogenic functions of tissue factor in humans. © 1998 by The American Society of Hematology.

The diagnosis of von Willebrand disease (VWD), the most common inherited bleeding disorder, is characterised by a variable bleeding tendency and heterogeneous laboratory phenotype. The sequencing of the entire VWF coding region has not yet become a routine practice in diagnostic laboratories owing to its high costs. Nevertheless, next-generation sequencing (NGS) has emerged as an alternative to overcome this limitation. We aimed to determine the correlation of genotype and phenotype in 92 Portuguese individuals from 60 unrelated families with VWD; therefore, we directly sequenced VWF. We compared the classical Sanger sequencing approach and NGS to assess the value-added effect on the analysis of the mutation distribution in different types of VWD. Sixty-two different VWF mutations were identified, 27 of which had not been previously described. NGS detected 26 additional mutations, contributing to a broad overview of the mutant alleles present in each VWD type. Twenty-nine probands (48.3 %) had two or more mutations; in addition, mutations with pleiotropic effects were detected, and NGS allowed an appropriate classification for seven of them. Furthermore, the differential diagnosis between VWD 2B and platelet type VWD (n = 1), Bernard-Soulier syndrome and VWD 2B (n = 1), and mild haemophilia A and VWD 2N (n = 2) was possible. NGS provided an efficient laboratory workflow for analysing VWF. These findings in our cohort of Portuguese patients support the proposal that improving VWD diagnosis strategies will enhance clinical and laboratory approaches, allowing to establish the most appropriate treatment for each patient.

The clinical courses of polycythemia vera (PV) and essential thrombocythemia (ET) are characterized by thrombohemorrhagic diathesis. Several groups have suggested an association between JAK2V617F mutation and thrombosis. We hypothesized a relationship between JAK2V617F allele burden, cellular activation parameters, and thrombosis. We evaluated a group of PV and ET patients using flow cytometry: platelet CD62P, CD63, and dense granules, platelet-leukocyte aggregates (PLA), leukocyte CD11b and monocyte tissue factor (TF) expression. All patients had increased baseline platelet CD62P and CD63 expression (p < 0.05); 71 % of PV and 47 % of ET presented with a storage pool disease. Leukocyte CD11b, TF, and PLA were elevated in all patients. TF was higher in PV compared to ET (p < 0.05) and platelet-neutrophil [polymorphonuclear (PMN)] aggregates were increased in ET versus PV (p < 0.05). In ET, PLA were correlated with platelet numbers (p < 0.05). In all patients, JAK2V617F allele burden was directly correlated with monocyte CD11b. Patients with JAK2V617F allele burden >50 % presented higher levels of leukocyte activation. In ET, thrombosis was associated with JAK2V617F mutation (p < 0.05, χ (2) = 5.2), increased monocyte CD11b (p < 0.05) and with platelet-PMN aggregates (p < 0.05). In ET patients, hydroxyurea does not significantly reduce the activation parameters. Our data demonstrate that JAK2V617F allele burden is directly correlated with activation parameters that drive mechanisms that favor thrombosis.