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- Base molecular da hemocromatose hereditária não-clássica em PortugalPublication . Faria, Ricardo; Silva, Bruno; Silva, Catarina; Loureiro, Pedro; Queiroz, Ana; Esteves, Jorge; Mendes, Diana; Fleming, Rita; Vieira, Luís; Gonçalves, João; Lavinha, João; Faustino, PaulaA Hemocromatose Hereditária (HH) é uma doença autossómica recessiva caracterizada pela absorção excessiva de ferro a nível intestinal e sua acumulação em órgãos vitais, podendo originar cardiomiopatia, cirrose e carcinoma hepatocelular. O correspondente diagnóstico molecular é obtido pela associação com genótipos específicos no gene HFE (homozigotia para p.Cys282Tyr ou heterozigotia composta p.Cys282Tyr/p.His63Asp). Contudo, nos países do sul da Europa, cerca de um terço dos doentes com diagnóstico clínico de HH não apresenta os referidos genótipos. Para identificar a base molecular da HH não-clássica em Portugal usaram-se metodologias de pesquisa geral de variantes genéticas (SSCP e dHPLC), Next-Generation Sequencing (NGS) e sequenciação de Sanger, cobrindo seis genes relacionados com o metabolismo do ferro em 303 doentes. Identificaram-se 69 variantes diferentes e de vários tipos, por ex. missense, nonsense, de splicing, que perturbam a transcrição do gene ou a regulação da tradução do mRNA. Seguidamente, realizaram-se estudos in silico e in vitro para esclarecer o significado etiológico de algumas das novas variantes. Concluiu-se que a base molecular desta patologia é bastante heterogénea e que a NGS é uma ferramenta adequada para efetuar a análise simultânea dos vários genes num grande número de amostras. Contudo, o estabelecimento da relevância clínica de algumas variantes requer a realização de estudos funcionais.
- Doença da Ferroportina - A propósito de um caso clínicoPublication . Novo, Elena; Faria, Ricardo; Faustino, Paula; Fleming, RitaIntrodução: A Hemocromatose Hereditária tipo 4 (HH-4), também designada Doença da Ferroportina é uma doença genética de sobrecarga em ferro devida a alteração da proteína ferroportina que tem como função a exportação de ferro das células e é alvo de regulação negativa pela hormona hepcidina. A patologia ocorre por mutações no gene SLC40A1, tem transmissão autossómica dominante, fenotipicamente heterogénea com 2 subtipos (forma A e B) e prevalência mundial <1/1.000.000. Objetivo: A HH-4 pertence ao grupo das Doenças Raras (cerca de 200 casos descritos mundialmente); procede-se à descrição do caso clínico de doente enviada à consulta de Imuno-hemoterapia por hiperferritinémia. Doente e Métodos: Doente do sexo feminino, 41 anos, em que foram estudadas causas de hiperferritinémia, nomeadamente síndroma metabólico, parâmetros inflamatórios, doença hepática crónica, hemólise e aceruloplasmina, cujos resultados foram negativos. Seguiu-se quantificação de ferro hepático por Ressonância Magnética (RM). Perante os resultados, fez-se pesquisa de mutações raras para HH. Iniciou-se Flebotomias.Terapêuticas (FT) de 450cc semanais que passaram a quinzenais para atingir ferritina <50ng/ml. A probanda refere que o pai e uma irmã fazem FT. No estudo genético do pai foi detetada uma mutação em heterozigotia no gene SLC40A1, o que direcionou o estudo genético na doente. Resultados (antes/após 4 FT): Ferritina: 708/553 ng/ml; Saturação transferrina: 27/30 %; ALT: 17/20 U/L;Glicemia: 88/- mg/dl; RM: 85/- μmol/g; Hemoglobina: 13,6/12,5 g/dl. Genética: A análise do gene SLC40A1 revelou heterozigotia para a mutação c.238G>A o que origina a alteração p.Gly80Ser na ferroportina, perda da sua função e acumulação intracelular de ferro. Houve necessidade de espaçar as FT, por rápida baixa de hemoglobina já prevista pela patologia, mantendo-se ainda em fase de indução. Discussão: Perante hiperferritinémia, após eliminar causas de Hemocromatose Secundária, havendo ligeira sobrecarga de ferro hepático e história familiar, diagnosticou-se um caso de Doença da Ferroportina, forma A. Embora a doente apresente um padrão benigno com saturação de transferrina normal e sobrecarga inicial essencialmente a nível das células de Kupffer, optou-se por iniciar FT dada a tendência para aumento de sobrecarga com a idade. l
- Ferroportin Disease (Haemochromatosis-type IV): a case reportPublication . Novo, Elena; Faria, Ricardo; Faustino, Paula; Fleming, RitaIntrodution: Haemochromatosis-type IV, the ferroportin disease, is characterized by an autosomal-dominant transmission and early iron accumulation in macrophages. It is caused by mutations in the transmembrane iron exporter protein ferroportin1 (SLC40A1 gene). In form A (classic), ferroportin loss of function mutants are unable to export iron from cells leading to cellular iron accumulation with decreased availability of iron for serum transferrin (TS). We present a Portuguese rare clinical case of HH-IV. Materials and Methods: A 41-year-old woman with hyperferritinemia and normal TS. Causes of hyperferritinemia (inflammation, chronic alcohol consumption, metabolic syndrome, cell necrosis, non-alcoholic fatty liver disease and aceruloplasminemia) were assessed. Liver iron, evaluated by magnetic resonance imaging (MRI) was carried out. Screening for mutation in HFE and SCL40A1 genes were performed by Sanger sequencing. Baseline: Ferritin:708ng/ml; TS: 27%; MRI:85µmol/g; Hb:13,6g/dl. Therapy: weekly 450ml Therapeutic Phlebotomies (TP) until ferritin≤50ng/ml. Results: Hyperferritinemia comorbidities and common genetic mutations for haemochromatosis were negative. However, sequencing of the patient SLC40A1 gene has revealed the presence in heterozygosity of the variant c.238G>A; p.Gly80Ser. Due to low tolerance to TP, we adopted smaller phlebotomies every three weeks. Conclusion: This patient has a rare autosomal-dominant Ferroportin disease due to a mutated ferroportin which is predicted to be defective in iron cellular export. In agreement, she presents hyperferritinemia, with normal TS and liver iron overload. The genotype/phenotype association allowed to diagnosis this rare FD case. Although a mild form A, we decided to start TP. Her father also has been treated for iron overload.
- Next Generation Sequencing of six iron-metabolism related genes in Portuguese patients with iron overload and a negative Hereditary Hemochromatosis-first level genetic test: a pilot studyPublication . Faria, Ricardo; Silva, Bruno; Silva, Catarina; Vieira, Luís; Loureiro, Pedro; Gomes, Susana; Gonçalves, João; Fleming, Rita; Faustino, PaulaIntroduction: Hereditary Hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive intestinal iron absorption and iron deposition in several organs leading to cardiac failure, cirrhosis and hepatocellular carcinoma. Molecular diagnosis of HFE-related HH is typically made by searching for specific genotypes (C282Y homozygosity or C282Y/H63D compound heterozygosity), denominated the “first level genetic test”. However, in the Mediterranean area, up to one third of patients with a clinical diagnosis of hemochromatosis do not present those mutations. This pilot study was designed to develop a “second level genetic test” based on next generation sequencing (NGS) for rapid and simultaneous analysis of 6 HH-related genes (HFE, TfR2, HJV, HAMP, SLC40A1 and FTL). A second objective was to establish genotype/phenotype associations. Patients and Methods: A TruSeq Custom Amplicon (TSCA, by Illumina) kit was designed in order to generate 97 amplicons covering exons, intron/exon junctions and UTRs of the mentioned genes with a cumulative target sequence of 12115bp. Amplicons were sequenced in the MiSeq instrument (IIlumina) using 250bp pared-end reads. Sequences were aligned against human genome reference hg19 using alignment and variant caller algorithms in the MiSeq reporter software. Firstly, some controls presenting known mutations were sequenced in order to validate the test. Subsequently, 88 iron overload patients with a negative first level test were studied according to previous conditions. Results: We found a total of 55 variants in the 6 selected genes. These include novel missense and splicing variants, a mutation that originates a novel translation initiation codon, among others. Novel potentially pathogenic variants were validated by Sanger sequencing and their functional significance are currently under study. An unusual clinical case will be presented. Discussion: The merger between TSCA methodology and NGS technology appears to be an appropriate tool for simultaneous and fast analysis of HH-related genes in a large number of samples. However, establishing the clinical relevance of NGS-detected variants for HH development remains a hard-working task, requiring further functional studies.
- Next-generation sequencing of hereditary hemochromatosis-related genes: novel likely pathogenic variants found in the Portuguese populationPublication . Faria, Ricardo; Silva, Bruno; Silva, Catarina; Loureiro, Pedro; Queiroz, Ana; Fraga, Sofia; Esteves, Jorge; Mendes, Diana; Fleming, Rita; Vieira, Luís; Gonçalves, João; Faustino, PaulaHereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive iron absorption resulting in pathologically increased body iron stores. It is typically associated with common HFE gene mutation (p.Cys282Tyr and p.His63Asp). However, in Southern European populations up to one third of HH patients do not carry the risk genotypes. This study aimed to explore the use of next-generation sequencing (NGS) technology to analyse a panel of iron metabolism-related genes (HFE, TFR2, HJV, HAMP, SLC40A1, and FTL) in 87 non-classic HH Portuguese patients. A total of 1241 genetic alterations were detected corresponding to 53 different variants, 13 of which were not described in the available public databases. Among them, five were predicted to be potentially pathogenic: three novel mutations in TFR2 [two missense (p.Leu750Pro and p.Ala777Val) and one intronic splicing mutation (c.967-1GNC)], one missense mutation in HFE (p.Tyr230Cys), and one mutation in the 5′-UTR of HAMP gene(c.-25GNA). The results reported here illustrate the usefulness of NGS for targeted iron metabolism-related gene panels, as a likely cost-effective approach for molecular genetics diagnosis of non-classic HH patients. Simultaneously, it has contributed to the knowledge of the pathophysiology of those rare iron metabolism-related disorders.
- Next-generation sequencing of iron-metabolism related genes in Portuguese patients with iron overload: novel pathogenic genetic variantsPublication . Faria, Ricardo; Silva, Bruno; Silva, Catarina; Vieira, Luis; Loureiro, Pedro; Gomes, Susana; Gonçalves, João; Rivera, Isabel; Fraga, Sofia; Fleming, Rita; Faustino, PaulaObjective: In Southern European countries up to one-third of the patients with hereditary hemochromatosis (HH) do not present the common HFE risk genotype. In order to investigate the molecular basis of these cases we have designed a gene panel for rapid and simultaneous analysis of 6 HH-related genes (HFE, TFR2, HJV, HAMP, SLC40A1 and FTL) by next-generation sequencing (NGS). Materials and Methods: Eighty-eight iron overload Portuguese patients, negative for the common HFE mutations, were analysed. A TruSeq Custom Amplicon kit (TSCA, by Illumina) was designed in order to generate 97 amplicons covering exons, intron/exon junctions and UTRs of the mentioned genes with a cumulative target sequence of 12115bp. Amplicons were sequenced in the MiSeq instrument (IIlumina) using 250bp paired-end reads. Sequences were aligned against human genome reference hg19 using alignment and variant caller algorithms in the MiSeq reporter software. Novel variants were validated by Sanger sequencing and their pathogenic significance were assessed by in silico studies. Results: We found a total of 55 different genetic variants. These include novel pathogenic missense and splicing variants (in HFE and TFR2), a very rare variant in IRE of FTL, a variant that originates a novel translation initiation codon in the HAMP gene, among others. Conclusion: The merging of TSCA methodology and NGS technology appears to be an appropriate tool for simultaneous and fast analysis of HH-related genes in a large number of samples. However, establishing the clinical relevance of NGS-detected variants for HH development remains a hard-working task, requiring further functional studies.
- Rare autosomal dominant hereditary hemochromatosis associated with SLC40A1 gene: ferroportin disease or type 4 hereditary hemochromatosis?Publication . Simão, Rita; Fleming, Rita; Mendonça, Joana; Machado, Miguel P.; Vieira, Luís; Tavares, Wilson; Lavinha, João; Faustino, PaulaFerroportin (FPN1), encoded by the SLC40A1 gene, is the unique cellular iron exporter identified in mammals. FPN1 transfers iron from the intestine and macrophages into the bloodstream. This function is negatively regulated by hepcidin. Mutations in SLC40A1 may affect FPN1 function, originating distinct autosomal dominant diseases: (i) the Ferroportin Disease (FD), due to loss-of-function mutations, is characterized by decreased iron export from enterocytes and severely affected iron transfer in macrophages, giving rise to a marked iron accumulation in spleen and liver; and (ii) the Type 4 Hereditary Hemochromatosis (HH), resulting from gain-of-function mutations conferring resistance to hepcidin-mediated FPN1 degradation and consequently high cellular iron export. In this study, 335 individuals suspected of having non-classic HH were enrolled. Six genes related with iron metabolism were analysed by SSCP, dHPLC or NGS. The latter used TruSeq or Nextera XT libraries and a MiSeq platform (Illumina). Genetic variants found were validated by Sanger sequencing. Predictive consequences at protein level were evaluated using Polyphen-2 and SIFT softwares. From all patients analysed, three SLC40A1 pathogenic variants were detected in heterozygosity in three women: two missense, c.238G>A, p.Gly80Ser and c.610G>A, p.Gly204Ser; and one deletion, c.485_487delTTG; p.Val162del. These variants had been reported in public databases, but they were not known to be present in the Portuguese population. The p.Gly80Ser and the p.Val162del are FPN1 loss-of-function mutations and were found associated with hyperferritinemia and low transferrin saturation (FD). In contrast, the p.Gly204Ser induced a gain of FPN1 function with a full iron export capacity giving the patient a type 4-HH phenotype, which includes iron overload, hyperferritinemia and high transferrin saturation. Detailed clinical evaluation of the suspected patients are useful to unravel the effect of different mutations in FPN1 function, expression and regulation.
- The functional significance of E277K and V295A HFE mutationsPublication . Silva, Bruno; Martins, Rute; Proença, Daniela; Fleming, Rita; Faustino, PaulaHereditary haemochromatosis (HH) is an autosomal recessive disorder characterized by excessive intestinal iron absorption resulting in increased pathological body iron stores. It is typically associated with homozygosity for the c.845G>A (p.C282Y) mutation in the HFE gene. However, other HFE alterations have been reported in affected individuals but their association with the disease is unclear. This study analysed the functional consequences of two HFE mutations, c.829G>A (p.E277K) and c.884T>C(p.V295A). Firstly, it was shown that c.829G>A affects the HFE splicing by diminishing the full length HFE and ivs4_66bp inclusion transcript levels, while increasing the amount of exon 4 skipping transcript. Immunofluorescent techniques showed that the HFE_E277K protein had a diffuse distribution(similar to HFE_C282Y) while HFE_V295A presented at the cell surface and perinuclear compartments (resembling HFE_wt). Immunoprecipitation assays revealed a decreased association of HFE_E277K and HFE_V295A with both b2-microglobulin (B2M; 38 ± 7% and 66 ± 8%, respectively) and transferrin receptor (TFRC, also termed TFR1) (58 ± 2% and 49 ± 16%, respectively). Herein, we prove that both mutations partially abrogate HFE association with B2M and TFRC, crucial for its correct processing and cell surface presentation. Although E277K has a more deleterious effect than V295A, we propose that both mutations may play a role in the development of hereditary haemochromatosis.