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Dissertação de mestrado em Biologia Humana e Ambiente, apresentada à Faculdade de Ciências da Universidade de Lisboa, 2020. This dissertation was conducted in the Department of Human Genetics, I&D Unit, Group of Hemoglobinopathies, Iron Metabolism and Associated Pathologies, at the National Institute of Health Doutor Ricardo Jorge (INSA), IP, Lisbon, Portugal. Dissertação de mestrado orientada por Doutora Paula Faustino (INSA) e Prof.ª Doutora Maria Teresa Rebelo (FCUL). A anemia é um problema de saúde pública global que afeta 24,8 % da população mundial, sendo maioritariamente causada por deficiências nutricionais de ferro. Para além disso, quando perturbados os mecanismos moleculares associados à homeostase do ferro é possível que surjam anemias hereditárias de difícil diagnóstico, como é o caso da Iron-Refractory Iron Deficiency Anemia (IRIDA). Por outro lado, alterações na síntese ou estrutura das cadeias globínicas originam também anemias hereditárias denominadas hemoglobinopatias. Primeiramente, de forma a explicar fenótipos atípicos de hemoglobinopatias e entender os mecanismos fisiopatológicos subjacentes aos mesmos, 15 casos clínicos de hemoglobinopatias foram estudados utilizando as metodologias PCR, gap-PCR, Multiplex Ligation-Dependent Probe Amplification (MLPA) e sequenciação automática de Sanger para analisar vários genes globínicos (HBB; HBD; HBG1; HBG2; HBA1; e HBA2). Este estudo permitiu o esclarecimento das causas moleculares associadas aos fenótipos complexos destes indivíduos, nos quais estavam presentes lesões moleculares responsáveis por: variantes estruturais raras de hemoglobina, talassémias do tipo alfa e beta e polimorfismos responsáveis pela modulação da expressão dos genes globínicos De seguida, o trabalho focou-se no estudo de 10 casos suspeitos de IRIDA, através da amplificação do gene TMPRSS6, que codifica a proteína matriptase-2, por PCR-longo e análise por Next-generation Sequencing, seguida de análise in silico, de forma a prever o possível efeito patogénico na proteína de variantes genéticas novas/raras. Recorreu-se à ferramenta bioinformática Human Splicing Finder de forma a detetar potenciais alterações no processo de splicing e às ferramentas PolyPhen-2, SIFT e Missense3D, a fim de analisar o impacto das variantes missense ao nível da estrutura e função da proteína codificada. Esta investigação permitiu apenas justificar o fenótipo de um dos casos em estudo, cujo quadro clínico julga-se ser fruto da herança digénica de uma nova mutação patogénica (TMPRSS6: c.871G>A; G291S) e 3 polimorfismos modeladores no mesmo gene (K253E, S361S e Y739Y) em conjunto com a co-herança do alelo para a deleção α-talassémica de 3,7 kb, que levam à presença de um fenótipo tipo-IRIDA. De forma a detetar a presença e entender o papel de polimorfismos comuns em genes relacionados com o metabolismo do ferro, foi realizado um estudo populacional de associação para investigar a influência que os polimorfismos nos genes TMPRSS6 (V736A e K253E) e HFE (H63D e C282Y) têm sobre os parâmetros hematológicos e do status do ferro de 317 indivíduos adultos, portugueses, divididos em 3 grupos: portadores de β-talassémia (BTT), indivíduos com anemia ferropénica (IDA) e controlos normais. Os indivíduos foram genotipados recorrendo às técnicas PCR-(RFLP) Restriction fragment length polymorphism e Amplification-Refractory Mutation System (ARMS)-PCR. Os resultados sugerem que ambos os genótipos heterozigótico (TC) e homozigótico (CC) da variante V736A exercem um efeito protetor contra a anemia ferropénica e que indivíduos controlo do sexo feminino com o genótipo CC têm os parâmetros volume globular médio (MCV), hemoglobina corpuscular média (MCH) e saturação de transferrina (TSAT) significativamente aumentados quando comparados com o genótipo wild-type (TT). Os resultados da análise de regressão logística detetaram uma associação positiva entre a presença da variante H63D e os níveis de dispersão do volume eritrocitário (RDW) nos controlos saudáveis e verificou-se uma associação entre esta mesma variante e os parâmetros do status do ferro de indivíduos BTT. Após o estudo de associação, foi testado o desempenho de diagnóstico de 13 índices matemáticos diferenciadores de anemia microcítica em indivíduos BTT ou IDA, com o intuito de determinar quais seriam os mais adequados à população Portuguesa. A análise revelou que para uma população adulta de mulheres portuguesas, os índices com melhor desempenho são RBC, G&K e RDWI. Os resultados foram ao encontro do reportado para outros países Mediterrânicos onde a β-talassémia é endémica. O mesmo verificou-se para o Brasil e França, provavelmente devido, respetivamente, à herança portuguesa e a fluxos migratórios. No geral, os resultados indicam que estes índices podem auxiliar no diagnóstico da causa subjacente à anemia microcítica, reconhecendo indivíduos suspeitos de BTT e encaminhando-os para testes bioquímicos e moleculares adicionais. Em suma, este trabalho permitiu entender melhor as alterações genéticas responsáveis pelas anemias hereditárias, bem como a forma como estas alteram o metabolismo do ferro, levando a um melhor conhecimento dos fatores que conduzem à variabilidade clínica em pacientes com estas doenças. Concluímos também que o estudo destas patologias tornou possível um correto diagnóstico de algum dos indivíduos estudados, permitindo um adequado acompanhamento clínico e aconselhamento genético, que leva por sua vez a uma melhoria da qualidade de vida dos casos em questão. Ademais, este trabalho reforça a importância do estudo dos marcadores moleculares e do uso de ferramentas não moleculares que complementam as técnicas de diagnóstico já implementadas. Anemia is a global public health problem affecting 24.8 % of the world population and is mostly caused by nutritional iron deficiencies. Additionally, molecular mechanism´s disturbances associated with the iron homeostasis may cause hereditary anemias of challenging diagnosis, such as Iron-Refractory Iron Deficiency anemia (IRIDA). Alternatively, globin chains structure or synthesis changes may lead to hemoglobinopathies, another type of hereditary anemia. Firstly, in order to explain atypical hemoglobinopathies’ phenotypes and understand underlying pathophysiological mechanisms, 15 hemoglobinopathies clinical cases were studied by PCR, gapPCR, Multiplex Ligation-Dependent Probe Amplification (MLPA) and automated Sanger sequencing of the various globin genes. These techniques made it possible to clarify the molecular causes behind these complex phenotypes, with molecular lesions being responsible for rare hemoglobin structural variants, β- and α-thalassemias, and the presence of polymorphisms that modulate the expression of globin genes. Secondly, we focused on ten IRIDA suspects, by amplifying their TMPRSS6 gene by long-PCR followed by Next-generation Sequencing and in silico analysis for predicting the possible pathogenic effect of the detected genetic variants. Through this analysis, only the phenotype of one patient could be justified by the presence of digenic inheritance of the novel damaging mutation (TMPRSS6: c.871G>A; G291S) and 3 common modulating SNPs in the same gene (K253E, S361S and Y739Y), in addition to the co-inheritance of the –α 3.7kb -thalassemia allele, which may add up to an IRIDA-like phenotype. Thirdly, to detect the presence and understand the roles and of common SNPs in genes related to iron metabolism, a case-control study was performed to investigate the influence of TMPRSS6 (V736A and K253E) and HFE (H63D and C282Y) on the hematological and iron parameters of 317 Portuguese subjects divided into 3 groups: β-thalassemia trait (BTT), iron deficiency anemia (IDA) and controls. Subjects were genotyped using PCR-(RFLP) Restriction fragment length polymorphism and Amplification-Refractory Mutation System (ARMS)-PCR techniques. Results suggests that both heterozygous (TC) and homozygous (CC) genotypes of V736A act as a protective factor against IDA, and that in the female control group, the CC genotype had significantly increased MCV, MCH and TSAT parameters when compared to the wild type (TT). Logistic regression results detected a positive association between H63D variant and RDW in control subjects, and an association between this variant and the iron parameters of BTT subjects. Finally, we evaluated the diagnostic performance of 13 mathematical indices for differentiating microcytic anemia in BTT and or IDA subjects, to find which ones would better apply to the Portuguese population. When accessing the performance of the indices we found that for a female adult Portuguese population the best performing ones where RBC, G&K and RDWI, similarly to other Mediterranean countries, and also France and Brazil, the latter probably resulting from migratory movements and Portuguese ancestry, respectively. Results imply that these indices can aid diagnosing the condition underlying microcytic anemia, recognizing individuals suspected of BTT and forwarding them to additional biochemical and molecular tests. The study of hereditary anemia made it possible for us to assign a correct diagnostic to the studied patients, allowing for correct clinical management and genetical counselling, that in turn, improves their quality of life. Additionally, this work has reinforced the importance of the study of molecular markers and use of non-molecular tools complementary to the already implemented diagnostic techniques. The work was partially funded by Fundação para a Ciência e a Tecnologia (FCT) ISAMB, and INSA projects 2012DGH720 and 2013DGH910. Additionally, it is a result of the GenomePT project (POCI-01-0145-FEDER022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), FCT. N/A

publicado em: Journal of Sickle cell Disease and Hemoglobinopathies. 2020 Jun 12;7:40. Sickle cell disease is a monogenic disease with early onset manifestations that begin in childhood and is characterized by high clinical heterogeneity, being influenced by genetic and environmental factors. Among disease modulators fetal hemoglobin and alpha thalassemia are among the most important. There is little data on these factors in sickle cell disease patients from Africa and none from Angola where the prevalence of the disease is very high. The aim of this study was to explore the possible association between alpha thalassemia, Fetal hemoglobin, hematological indices and clinical events in Angolan sickle cell disease Hydroxyurea-naïve pediatric patients. This cross-sectional study is part of a large study in an Angolan sickle cell disease cohort conducted in the Hospital Pediátrico David Bernardino in Luanda and in Hospital Geral do Bengo in Caxito. Sampling was performed between April and August 2019. A total of 200 sickle cell disease children were included, after guardian informed consent, being 51,5% females. A venous blood sample was collected from each participant and used for hematological analyses, electrophoresis for diagnosis confirmation, Fetal hemoglobin quantification by HPLC (Bio-Rad variant II). DNA isolation was done by Qiagen blood mini kit, and the 3.7 kb alpha thalassemia deletion was studied by GAP-PCR. ANOVA, non-parametric tests and Chi-square tests were applied to compare the means, medians or frequencies between the three alpha thalassemia genotypes. P-value

© Springer Nature B.V. 2020, The aim of this study was to explore the association between alpha-thalassemia, fetal hemoglobin, hematological indices, and clinical adverse events in Angolan sickle cell disease pediatric patients. A total of 200 sickle cell disease (SCD) children were sampled in Luanda and Caxito. A venous blood sample was collected and used for hematological analyses, fetal hemoglobin quantifcation, and genotyping of 3.7 kb alpha-thalassemia deletion by GAP-PCR. The frequency of the 3.7 kb alpha-thalassemia deletion in homozygosity was 12.5% and in heterozygosity was 55.0%. An increase in alpha-thalassemia frequency was observed in children older than 5 years old (11.7% vs. 13.00%). Furthermore, 3.7 kb alpha-thalassemia deletion homozygotes had a signifcantly higher age of the frst manifestation, lower number of blood transfusions by year, higher hemoglobin, lower mean corpuscular volume, mean corpuscular hemoglobin, and lower hemolytic rate observed by a lower number of reticulocytes count. There were no diferences in fetal hemoglobin between the three genotypes. Moreover, the number of stroke events, osteomyelitis, splenomegaly, splenectomy, and hepatomegaly were lower when alpha-thalassemia was co-inherited. For the frst time in Angolan population, the efect of alpha-thalassemia deletion in sickle cell disease was analyzed and results reinforce that this trait infuences the hematological and clinical aspects and produces a milder phenotype., This work was supported by the Fundação Para a Ciência e Tecnologia I.P./Aga Khan Development Network Project Number 330842553.