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- A novel haemoglobin variant mimicking cyanotic congenital heart diseasePublication . Abecasis, F; Marques, I; Bento, C; Ferrão, AScreening for critical congenital heart defects in newborn babies can aid in early recognition, with the prospect of improved outcome. However, as this universal newborn screening is implemented, there will be an increasing number of false-positive results. In order to avoid multiple investigations and uncertainty, an haemoglobin (Hb) variant must be included in the differential diagnosis in otherwise well newborns with low oxygen saturation by pulse oximetry. We describe a novel fetal Hb variant (heterozygous γ-globin gene (HBG1) mutation in exon 2 c.202G>A (p.Val68Met)) identified in a newborn with positive pulse oximetry screening for congenital heart disease.
- Aberrant p15, p16, p53, and DAPK Gene Methylation in Myelomagenesis: Clinical and Prognostic ImplicationsPublication . Geraldes, C; Gonçalves, AC; Cortesão, E; Pereira, MI; Roque, A; Paiva, A; Ribeiro, L; Nascimento-Costa, JM; Sarmento-Ribeiro, ABBACKGROUND: Aberrant DNA methylation is considered a crucial mechanism in the pathogenesis of monoclonal gammopathies. We aimed to investigate the contribution of hypermethylation of 4 tumor suppressor genes to the multistep process of myelomagenesis. METHODS: The methylation status of p15, p16, p53, and DAPK genes was evaluated in bone marrow samples from 94 patients at diagnosis: monoclonal gammopathy of uncertain significance (MGUS) (n = 48), smoldering multiple myeloma (SMM) (n = 8) and symptomatic multiple myeloma (MM) (n = 38), and from 8 healthy controls by methylation-specific polymerase chain reaction analysis. RESULTS: Overall, 63% of patients with MM and 39% of patients with MGUS presented at least 1 hypermethylated gene (P < .05). No aberrant methylation was detected in normal bone marrow. The frequency of methylation for individual genes in patients with MGUS, SMM, and MM was p15, 15%, 50%, 21%; p16, 15%, 13%, 32%; p53, 2%, 12,5%, 5%, and DAPK, 19%, 25%, 39%, respectively (P < .05). No correlation was found between aberrant methylation and immunophenotypic markers, cytogenetic features, progression-free survival, and overall survival in patients with MM. CONCLUSIONS: The current study supports a relevant role for p15, p16, and DAPK hypermethylation in the genesis of the plasma cell neoplasm. DAPK hypermethylation also might be an important step in the progression from MGUS to MM.
- 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.
- JAK2V617F allele burden is associated with thrombotic mechanisms activation in polycythemia vera and essential thrombocythemia patientsPublication . Coucelo, M; Caetano, G; Sevivas, T; Almeida Santos, S; Fidalgo, T; Bento, C; Fortuna, M; Duarte, M; Menezes, C; Ribeiro, MLThe 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.
- Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNAPublication . Borràs, N; Orriols, G; Batlle, J; Pérez-Rodríguez, A; Fidalgo, T; Martinho, P; López-Fernández, MF; Rodríguez-Trillo, Á; Lourés, E; Parra, R; Altisent, C; Cid, AR; Bonanad, S; Cabrera, N; Moret, A; Mingot-Castellano, ME; Navarro, N; Pérez-Montes, R; Marcellini, S; Moreto, A; Herrero, S; Soto, I; Fernández-Mosteirín, N; Jiménez-Yuste, V; Alonso, N; de Andrés-Jacob, A; Fontanes, E; Campos, R; Paloma, MJ; Bermejo, N; Berrueco, R; Mateo, J; Arribalzaga, K; Marco, P; Palomo, Á; Castro Quismondo, N; Iñigo, B; Nieto, MM; Vidal, R; Martínez, MP; Aguinaco, R; Tenorio, JM; Ferreiro, M; García-Frade, J; Rodríguez-Huerta, AM; Cuesta, J; Rodríguez-González, R; García-Candel, F; Dobón, M; Aguilar, C; Vidal, F; Corrales, ILarge studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7473G>A (p.=) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of 3 mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that 4 of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease.
- Atypical adult-onset methylmalonic acidemia and homocystinuria presenting as hemolytic uremic syndromePublication . Navarro, D; Azevedo, A; Sequeira, S; Ferreira, AC; Carvalho, F; Fidalgo, T; Vilarinho, L; Santos, MC; Calado, J; Nolasco, FThrombotic microangiopathy (TMA) syndromes can be secondary to a multitude of different diseases. Most can be identified with a systematic approach and, when excluded, TMA is generally attributed to a dysregulation in the activity of the complement alternative pathways-atypical hemolytic uremic syndrome (aHUS). We present a challenging case of a 19-year-old woman who presented with thrombotic microangiopathy, which was found to be caused by methylmalonic acidemia and homocystinuria, a rare vitamin B12 metabolism deficiency. To our knowledge, this is the first time that an adult-onset methylmalonic acidemia and homocystinuria presents as TMA preceding CNS involvement.
- New combined CFH/MCP mutations and a rare clinical course in atypical haemolytic uraemic syndromePublication . Lopes, D; Gomes, AM; Cunha, C; Pinto, CS; Fidalgo, T; Fernandes, JCAtypical haemolytic uraemic syndrome (aHUS) is a rare, life-threatening, chronic, genetic disease due to uncontrolled alternative pathway complement activation. In this report, we discuss the case of a heterozygous carrier of a mutation on both factor H and membrane cofactor protein, who persistently presents haemolytic anaemia without need for blood transfusions, normal platelet count, normal renal function and no signs or symptoms of organ injury due to thrombotic microangiopathy 4 years after the diagnosis of aHUS.
- 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.
- Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem CellsPublication . Garate, Z; Quintana-Bustamante, O; Crane, AM; Olivier, E; Poirot, L; Galetto, R; Kosinski, P; Hill, C; Kung, C; Agirre, X; Orman, I; Cerrato, L; Alberquilla, O; Rodriguez-Fornes, F; Fusaki, N; Garcia-Sanchez, F; Maia, TM; Ribeiro, ML; Sevilla, J; Prosper, F; Jin, S; Mountford, J; Guenechea, G; Gouble, A; Bueren, JA; Davis, BR; Segovia, JCPyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.
- [Hodgkin's Lymphoma and Autoimmunity: Is There a Relationship?]Publication . Jerónimo, M; Silva, S; Benedito, M; Brito, MJINTRODUCTION: The relationship between lymphomas and autoimmune diseases has been reported as bi-directional, however there is a few data in pediatric population. The aim of this work is to evaluate the prevalence of autoimmune diseases in children and adolescents with Hodgkin's lymphoma followed in a Pediatric Oncology Unit. MATERIAL AND METHODS: By reviewing Hodgkin's lymphomas data from the past 16 years (collected prospectively), an apparently large incidence of autoimmune diseases, mostly in female patients, was noted. We decided to do this retrospective study with an update on follow-up. Data analyzed: age, gender, autoimmune disease, temporal relation with lymphoma, lymphoma's stage, histologic subtype and treatment. RESULTS: Fifty-two cases were included, 7 (13.5%) of which, all female, had an autoimmune disease diagnosed previously, simultaneously or after lymphoma. Autoimmune diseases were: juvenile idiopathic arthritis, inflammatory bowel disease, Behçet's disease, autoimmune hepatitis, systemic erythematosus lupus, Hashimoto's thyroiditis and idiopathic thrombocytopenic purpura. The diagnosis was made after lymphoma in 4 patients, before in 2 and simultaneously in 1 patient. All cases, except the one with simultaneous diagnosis, are out of treatment and without oncologic disease relapse. No deaths were registered. DISCUSSION: There was an important prevalence of autoimmune diseases in girls with Hodgkin's lymphoma. We present data and discuss the possible causes based on a literature review. CONCLUSIONS: This relationship should be invoked, requiring more studies, especially in pediatric age.