Browsing by Author "Salvado, R"
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- Combined study of ADAMTS13 and complement genes in the diagnosis of thrombotic microangiopathies using next-generation sequencingPublication . Fidalgo, T; Martinho, P; Pinto, CS; Oliveira, AC; Salvado, R; Borràs, N; Coucelo, M; Manco, L; Maia, T; Mendes, MJ; Del Orbe Barreto, R; Corrales, I; Vidal, F; Ribeiro, MLBACKGROUND: The 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 METHODS: We 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. PATIENTS: For 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. RESULTS: In 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. CONCLUSIONS: Our 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.
- Familial thrombotic risk based on the genetic background of Protein C Deficiency in a Portuguese StudyPublication . Fidalgo, T; Martinho, P; Salvado, R; Manco, L; Oliveira, AC; Pinto, CS; Gonçalves, E; Marques, D; Sevivas, T; Martins, N; Ribeiro, MLINTRODUCTION: Inherited protein C (PC) deficiency is a well-known risk factor for venous thrombosis (VT). Plasma PC levels are reliable in moderate to severe deficiencies; however, in mildly deficient individuals, the levels may overlap with those considered normal. Genetic studies of PROC, which encodes PC, could help identify carriers; genome-wide association studies (GWAS) have shown that approximately 50% of phenotypic variation in PC deficiency is caused by the cumulative effects of mutations in several other loci, namely in the PROCR. PATIENTS AND METHODS: With the main objective of determining the genotype/phenotype correlation in 59 Portuguese individuals from 26 unrelated families with history of thrombosis and repeatedly low/borderline PC plasma levels, we conducted a molecular study by direct sequencing of PROC; PROC promoter haplotypes and PROCR c.4600A>G polymorphism (rs867186), which are known to influence plasma PC concentrations, were also screened. RESULTS: Twelve different PROC mutations were identified, one of them not previously reported, p.Cys105Arg. The mutation types and locations as well as haplotype combinations correlated with the phenotypic severity. The most frequent mutation, p.Arg199X, correlated with the CGTC haplotype and was identified in nine families containing patients with higher numbers of VT episodes. This mutation in homozygous individuals for the CGTC haplotype is a significant risk factor for VT in Portuguese. CONCLUSION: These genetic family studies allowed the identification of the unknown carriers and individuals at a higher thrombotic risk within each family, thus permitting the evaluation of the need for prophylactic measures, particularly in at-risk situations.
- Genotype-phenotype correlation in a cohort of Portuguese patients comprising the entire spectrum of VWD types: impact of NGSPublication . Fidalgo, T; Salvado, R; Corrales, I; Pinto, SC; Borràs, N; Oliveira, A; Martinho, P; Ferreira, G; Almeida, H; Oliveira, C; Marques, D; Gonçalves, Elsa; Diniz, MJ; Antunes, M; Tavares, A; Caetano, G; Kjöllerström, P; Maia, R; Sevivas, TS; Vidal, F; Ribeiro, LThe 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.
- Genotype-phenotype correlation in a cohort of Portuguese patients comprising the entire spectrum of VWD types: impact of NGSPublication . Fidalgo, T; Salvado, R; Corrales, I; Pinto, SC; Borràs, N; Oliveira, A; Martinho, P; Ferreira, G; Almeida, H; Oliveira, C; Marques, D; Gonçalves, E; Diniz, MJ; Antunes, M; Tavares, A; Caetano, G; Kjöllerström, P; Maia, R; Sevivas, T; Vidal, F; Ribeiro, LThe 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.
- Severe intracranial haemorrhage in neonatal alloimmune thrombocytopeniaPublication . Silva, F; Morais, S; Sevivas, T; Veiga, R; Salvado, R; Taborda, ANeonatal alloimmune thrombocytopenia is a rare (1/1000-5000 births) life-threatening disorder, caused by fetomaternal incompatibility for a fetal human platelet alloantigen inherited from the father, with production of maternal alloantibodies against fetal platelets, leading to severe thrombocytopenia and potential bleeding. Intracranial haemorrhage is the most feared complication. This report presents the case of a term newborn infant, born from caesarean section after a normal pregnancy, presenting signs of skin bleeding with different ages. Obstetric history included a previous spontaneous abortion after amniocentesis. Severe thrombocytopenia (4×10(9)/l platelets) was found and brain ultrasound showed multiple intracranial haemorrhages. Human platelet antigen (HPA) phenotyping showed maternal negative HPA-1a and paternal positive HPA-1a platelets. Strongly positive anti-HPA-1a and weakly positive anti-human leukocyte antigen class I alloantibodies were found in the mother. Multiple platelet transfusions, intravenous immunoglobulin and corticosteroid were given but favourable response was accomplished only after a compatible platelet transfusion. Brain MRI showed multiple subacute and chronic haemorrhages.