Leticia Ribeiro

Genotype-phenotype correlation in a cohort of Portuguese patients comprising the entire spectrum of VWD types: impact of NGS

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.

Nonsense-mediated mRNA decay (NMD) blockage promotes nonsense mRNA stabilization in protein 4.1R deficient cells carrying the 4.1R Coimbra variant of hereditary elliptocytosis

We describe a new approach to stabilize nonsense mRNA, based on the inhibition of the NMD mechanism, by combining cycloheximide-mediated inhibition of translation, and caffeine-mediated inhibition of UPF1 phosphorylation. This approach aimed to identify the impact of a 4.1R splicing mutation. This mutation is involved in a partial deficiency of 4.1R in the homozygous state in a patient with hereditary elliptocytosis and a moderated hemolytic anemia. We show that, in addition to two known minor shortened and stable spliceoforms, the mutation activates an intronic cryptic splice site, which results in a nonsense mRNA major isoform, targeted to degradation in intact cells by NMD. This accounts for the main cause of 4.1R partial deficiency. In a general perspective, blocking the NMD mechanism would help to identify a missing isoform, and pave the path for a molecular targeting strategy to circumvent a deleterious splicing pathway in favor of a therapeutic splicing pathway.

Clonal shifts in MDS – from SF3B1 to EZH2

Andr e Barbosa Ribeiro , Margarida Coucelo , Rita Tenreiro, Ana Teresa Sim~ oes, Gilberto Marques, Let ıcia Ribeiro , Em ılia Cortes~ao and Ana Bela Sarmento-Ribeiro Department of Clinical Hematology, Centro Hospitalar e Universit ario de Coimbra, Coimbra, Portugal; Centre of Investigation in Environment, Genetics and Oncobiology – CIMAGO, Coimbra, Portugal; Laboratory of Oncobiology and Hematology, University Clinic of Hematology and Applied Molecular Biology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; Department of Clinical Pathology, Centro Hospitalar e Universit ario de Coimbra, Coimbra, Portugal

Severe immune thrombocytopenia in pregnancy treated with Eltrombopag – A case report

Immune thrombocytopenia (ITP) during pregnancy is an acquired autoimmune disease present in 1-2 of every 1000 pregnancies. Thrombopoietin (TPO)-mimetic drugs, such as eltrombopag, have been successfully used for treatment of ITP during pregnancy, but studies regarding its safety during gestation are lacking. A 33-year-old nulliparous woman with a history of chronic ITP, presented at the emergency department with petechiae, epistaxis, bruises, conjunctival effusions and a platelet count of 3×109/L at 25 weeks gestation. Her pregnancy had been uneventful until then. She was unresponsive to a therapeutic escalade of corticosteroids, azathioprine and intravenous immunoglobulin (IV Ig) so, at 27 weeks, eltrombopag was initiated, and analytical and clinical improvement was achieved. Labor was induced at 37 weeks due to preeclampsia, culminating in a vacuum-assisted vaginal delivery. A healthy female newborn weighing 2400g was born. After delivery, both had normal platelet counts and remained clinically stable through follow-up.

Quantification and phenotypic characterization of peripheral blood Vδ1 + T cells in chronic lymphocytic leukemia and monoclonal B cell lymphocytosis: Quantification and phenotypic characterization of Vδ1 + T Cells

Vδ1+ T cells, a subset of γδ T cells, are responsible for innate‐like immune responses. Recently, an anti‐tumor function mediated by MHC‐unrestricted recognition of lipid and stress molecules, has also been described in these cells. This study aimed to quantify and phenotypically characterize circulating Vδ1+ T cells in B cell Chronic Lymphocytic Leukemia (CLL) and Monoclonal B cell lymphocytosis (MBL).