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.

Essentials The differential diagnosis of acute thrombotic microangiopathy (TMA) is challenging.To the ADAMTS13 activity < or >10% was added a next‐generation sequencing (NGS) gene panel.The ADAMTS13 mutation p.Cys754Arg was frequent in hereditary thrombotic thrombocytopenic purpura.We identified novel complement gene mutations and this procedure improved our diagnostic strategy. BackgroundThe 2 main forms of thrombotic microangiopathy (TMA) are thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS). Deficiency of ADAMTS13 and dysregulation of the complement pathway result in TTP and aHUS, respectively; however, overlap of their clinical characteristics makes differential diagnosis challenging.Objectives and MethodsWe aimed to develop a TMA diagnosis workflow based on ADAMTS13 activity and screening of ADAMTS13 and complement genes using a custom next‐generation sequencing (NGS) gene panel.PatientsFor this, from a cohort of 154 Portuguese patients with acute TMA, the genotype‐phenotype correlations were analyzed in 7 hereditary TTP (ADAMTS13 activity <10%, no inhibitor), 36 acquired TTP (ADAMTS13 activity <10%, presence of an inhibitor), and in 34 presumable aHUS.ResultsIn total, 37 different rare variants, 8 of which novel (in ADAMTS13,CFH, and CD46), were identified across 7 genes. Thirteen TTP patients were homozygous (n=6), compound heterozygous (n=2), and heterozygous (n=5) for 11 ADAMTS13 variants (6 pathogenic mutations). Among the 34 aHUS patients, 17 were heterozygous for 23 variants in the different complement genes with distinct consequences, ranging from single pathogenic mutations associated with complete disease penetrance to benign variants that cause aHUS only when combined with other variants and/or CFH and CD46 risk haplotypes or CFHR1‐3 deletion.ConclusionsOur study provides evidence of the usefulness of the NGS panel as an excellent technology that enables more rapid diagnosis of TMA, and is a valuable asset in clinical practice to discriminate between TTP and aHUS.

Background Haemophilia A is a hereditary bleeding disorder, which has been considered rare and chronic. The burden of this disease in Portugal remains unknown. The aim of this study was to estimate the annualized cost and health burden of haemophilia A in Portugal. Methods Data were extracted from a Portuguese expert panel, from official data and national literature. Annual costs were calculated from the perspective of the society including direct and indirect costs. Unitary costs were extracted from 2017 national official sources and are expressed in euros. Health burden was expressed in disability adjusted life years (DALYs) based on incidence and quality of life questionnaires. Estimates are presented for the overall population and stratified by severity, age group (< 18 years vs. adults) and inhibitor status. Results The yearly average cost per patient is estimated to range from €39,654/patient without inhibitors and €302,189/patient with inhibitors, representing a 7.6 fold difference. Amongst patients without inhibitors, the annual average cost was €401 in mild, €5327 in moderate and €85,805 in severe disease. Average cost per child and adult is €72,287 and €51,737, respectively. Direct costs represent approximately 95% of all costs, of which almost the totality accounts for clotting factor replacement therapy and bypassing agents. The total annual cost of haemophilia A for the Portuguese society was estimated to be €42,66 million, one third of which was related to the treatment of patients with inhibitors. It is estimated that haemophilia A is responsible for 3878 DALYs in Portugal (497 DALYs in mild, 524 DALYs in moderate, 2031 DALYs in severe patients without inhibitors and 784 DALYs in patients with inhibitors) for the cohort of 2017 (750 patients) or 5.2 DALY/patient during lifetime. Conclusions Despite being rare, the economic and health burden of haemophilia A is remarkable. The main cost driver is clotting factor replacement therapy. Moreover, haemophilia A is more costly in children than in adults and rises exponentially with disease severity. Electronic supplementary material The online version of this article (10.1186/s13023-019-1175-5) contains supplementary material, which is available to authorized users.

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Thrombocytopenia-absent radius (TAR) syndrome is a rare congenital disorder characterized by the bilateral absence of the radius and thrombocytopenia, and sometimes by other skeletal, gastrointestinal, cardiac, and renal abnormalities. The underlying genetic defect is usually the compound inheritance of a microdeletion in 1q21.1 (null allele) and a low-frequency, non-coding single nucleotide variant (SNV) in the RBM8A gene (hypomorphic allele). We report three new cases from two unrelated families. The two siblings presented the common genotype, namely the compound heterozygosity for a 1q21.1 microdeletion and the hypomorphic SNV c.-21G>A in RBM8A, whereas the third, unrelated patient presented a rare genotype comprised by two RBM8A variants: c.-21G>A (hypomorphic allele) and a novel pathogenic variant, c.343-2A>G (null allele). Of the eight documented RBM8A variants identified in TAR syndrome patients, four have hypomorphic expression and four behave as null alleles. The present report expands the RBM8A null allele spectrum and corroborates the particularities of RBM8A involvement in TAR syndrome pathogenesis.