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As hemoglobinopatias consistem num grupo heterogéneo de doenças genéticas, tendo uma elevada prevalência global, particularmente em África, destacando-se a Anemia das Células Falciformes (ACF) e as Talassemias. Em Angola, apesar de ser conhecida a elevada prevalência de ACF, a diversidade genética das variantes de hemoglobina permanece pouco caracterizada, limitando o desenvolvimento de estratégias de rastreio e de intervenção médica. Tem sido, ao longo dos anos, comprovado o importante papel do rastreio neonatal na redução da mortalidade infantil associada a hemoglobinopatias. Esta dissertação teve como objetivo caracterizar molecularmente as variantes de hemoglobina detetadas durante um Programa de Rastreio Neonatal de ACF, contribuindo para o conhecimento epidemiológico no país. Foram analisadas por Focagem Isoelétrica (IEF) 13 256 amostras de recém-nascidos, tendo-se selecionado 45 dessas amostras, que demonstraram perfis eletroforéticos atípicos, para análises moleculares complementares, nomeadamente PCR-RFLP, Gap-PCR e sequenciação de Sanger. Foram confirmadas variantes estruturais de elevada prevalência, como HbS e HbC, bem como variantes menos expectáveis, como HbE, sugerindo fenómenos de ancestralidade ou migração. Os resultados revelaram que 26,7% das amostras atípicas apresentavam genótipo AS, 17,8% genótipo AC, 2,2% genótipo AE e 2,2% genótipo SS, permanecendo 51,1% inconclusivas, sobretudo devido a limitações técnicas, fraca qualidade do DNA extraído de sangue seco e dificuldades na amplificação dos genes HBA. Foram ainda detetados SNPs reportados como benignos (rs10768683 e rs713040), mas cuja relevância clínica demonstra carecer de investigação adicional. Este estudo reforça a importância da combinação de métodos fenotípicos e moleculares para a confirmação diagnóstica de hemoglobinopatias, especialmente em contextos de elevada diversidade genética. A identificação de variantes inesperadas sublinha a necessidade de abordagens diagnósticas mais abrangentes, contemplando fenómenos migratórios e uma gama mais alargada de hemoglobinopatias. Os dados obtidos fornecem suporte científico para a consolidação e otimização de programas de rastreio neonatal em Angola, com potencial aplicabilidade em outros contextos africanos.
Abstract Hemoglobinopathies comprise a heterogeneous group of genetic disorders with high global prevalence, particularly in Africa, with Sickle Cell Disease (SCD) and Thalassemias standing out. In Angola, despite the well-known high prevalence of SCD, the genetic diversity of hemoglobin variants remains poorly characterized, limiting the development of screening strategies and medical interventions. Over the years, the important role of neonatal screening in reducing infant mortality associated with hemoglobinopathies has been demonstrated. This dissertation aimed to molecularly characterize hemoglobin variants detected during a Neonatal Screening Program for SCD, contributing to the epidemiological knowledge in the country. A total of 13,256 newborn samples were analyzed by Isoelectric Focusing (IEF), from which 45 samples showing atypical electrophoretic profiles were selected for complementary molecular analyses, namely PCR-RFLP, Gap-PCR, and Sanger sequencing. Structural variants of high prevalence, such as HbS and HbC, were confirmed, as well as less expected variants, such as HbE, suggesting phenomena of ancestry or migration. Results revealed that 26.7% of atypical samples presented the AS genotype, 17.8% the AC genotype, 2.2% the AE genotype, and 2.2% the SS genotype, while 51.1% remained inconclusive, mainly due to technical limitations, poor quality of DNA extracted from dried blood spots, and difficulties in amplifying the HBA genes. SNPs reported as benign (rs10768683 and rs713040) were also detected, though their clinical relevance still requires further investigation. This study reinforces the importance of combining phenotypic and molecular methods for the diagnostic confirmation of hemoglobinopathies, especially in contexts of high genetic diversity. The identification of unexpected variants highlights the need for broader diagnostic approaches, considering migratory phenomena and a wider range of hemoglobinopathies. The data obtained provide scientific support for the consolidation and optimization of neonatal screening programs in Angola, with potential applicability in other African contexts.
Abstract Hemoglobinopathies comprise a heterogeneous group of genetic disorders with high global prevalence, particularly in Africa, with Sickle Cell Disease (SCD) and Thalassemias standing out. In Angola, despite the well-known high prevalence of SCD, the genetic diversity of hemoglobin variants remains poorly characterized, limiting the development of screening strategies and medical interventions. Over the years, the important role of neonatal screening in reducing infant mortality associated with hemoglobinopathies has been demonstrated. This dissertation aimed to molecularly characterize hemoglobin variants detected during a Neonatal Screening Program for SCD, contributing to the epidemiological knowledge in the country. A total of 13,256 newborn samples were analyzed by Isoelectric Focusing (IEF), from which 45 samples showing atypical electrophoretic profiles were selected for complementary molecular analyses, namely PCR-RFLP, Gap-PCR, and Sanger sequencing. Structural variants of high prevalence, such as HbS and HbC, were confirmed, as well as less expected variants, such as HbE, suggesting phenomena of ancestry or migration. Results revealed that 26.7% of atypical samples presented the AS genotype, 17.8% the AC genotype, 2.2% the AE genotype, and 2.2% the SS genotype, while 51.1% remained inconclusive, mainly due to technical limitations, poor quality of DNA extracted from dried blood spots, and difficulties in amplifying the HBA genes. SNPs reported as benign (rs10768683 and rs713040) were also detected, though their clinical relevance still requires further investigation. This study reinforces the importance of combining phenotypic and molecular methods for the diagnostic confirmation of hemoglobinopathies, especially in contexts of high genetic diversity. The identification of unexpected variants highlights the need for broader diagnostic approaches, considering migratory phenomena and a wider range of hemoglobinopathies. The data obtained provide scientific support for the consolidation and optimization of neonatal screening programs in Angola, with potential applicability in other African contexts.
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Hemoglobinopatias Anemia das células falciformes Rastreio neonatal África talassemias Hemoglobinopathies Sickle cell disease Neonatal screening Africa Thalassemias IPL/IDI&CA2024/GenFalci_ESTeSL