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A hemoglobina fetal como fator modificador da gravidade clínica da anemia das células falciformes: o exemplo dos acidentes vasculares cerebrais
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Abstract(s)
A anemia das células falciformes (SCA) é uma anemia hemolítica hereditária de transmissão autossómica recessiva devida à mutação HBB:c.20A>T no gene da β-globina que dá origem à hemoglobina S (HbS). A HbS quando em desoxigenação polimeriza no glóbulo vermelho, conferindo-lhe a forma de foice e tornando-o rígido e frágil. A SCA caracteriza-se por manifestações clínicas de gravidade variável que podem incluir, por ex., hemólise crónica, crises vaso-oclusivas e acidente vascular cerebral (AVC). A variabilidade fenotípica é devida a fatores ambientais e genéticos, e o nível de hemoglobina fetal (Hb F) é considerado o principal modulador da doença.
Neste trabalho pretendeu-se investigar em crianças com SCA se o nível de HbF é modulado pelo seus genótipos em regiões polimórficas conhecidas nos genes HBG2, BCL11A, HBS1L-MYB e KLF1 e, por outro lado, se o nível de HbF está relacionado com o grau de hemólise que apresentam e o risco de desenvolvimento de AVC.
Foram estudadas 67 crianças com SCA de origem Africana. Estas foram agrupadas de acordo com o grau de vasculopatia cerebral (Controlo, Risco e AVC). Para o estudo molecular foi usada a PCR-RFLP, sequenciação de Sanger e sequenciação de nova geração. Efetuaram-se estudos in silico e análise estatística em SPSS.
Os resultados permitiram concluir que um nível baixo de HbF é um fator de risco para o desenvolvimento de AVC (p=0,005). Observou-se também que os doentes com os genótipos mais raros em HBG2 (rs7482144_TT+TC) apresentam níveis mais elevados de HbF (p=0,031). No gene BCL11A concluiu-se que o genótipo rs11886868_CC e o rs4671393_alelo_A apresentam-se tendencialmente associados a níveis mais elevados de HbF e, pelo contrário, os polimorfismos estudados em HBS1L-MYB não revelaram associação com os níveis de HbF. A análise do gene KLF1 revelou que 82,8% dos doentes possuía pelo menos uma variante neste gene, foram detetadas 11 variantes diferentes sendo uma delas não descrita, a Q342H, mas de efeito benigno na proteína.
Concluiu-se que é de grande interesse considerar o efeito conjunto das referidas variantes nos genes analisados uma vez que a combinação no indivíduo das variantes mais raras versus as mais comuns está relacionada com os níveis de HbF (p=0,021) constituindo portanto um perfil genético de valor prognóstico. Os fatores genéticos analisados mostraram ser importantes modificadores dos níveis de HbF e poderão vir a constituir alvos de abordagem terapêutica.
Sickle Cell Anemia (SCA) is a hereditary hemolytic anemia with autosomal recessive transmission caused by the HBB:c.20A>T mutation in the β-globin gene, giving rise to hemoglobin S (HbS). Deoxygenated HbS polymerizes in the red cell, which acquires a sickle shape and becomes rigid and fragile. SCA is characterized by a variable clinical severity, including chronic hemolysis, vaso-occlusive episodes, and ultimately stroke. This phenotypic variability is due to environmental and genetic factors, and level of fetal hemoglobin (Hb F) has been proposed as the more important disease modulator. In this work, we aimed to investigate if the level of HbF in children with SCA is modulated by their genotypes in known polymorphic regions in the genes HBG2, BCL11A, HBS1L-MYB and KLF1. We also aimed to investigate whether the level of HbF in these children is related with the degree of hemolysis and the risk of stroke. We have studied 67 children with SCA, of African origin. These children were grouped in accordance with their degree of cerebral vasculopathy (Control, Risk and Stroke). For the molecular analysis, methodologies such as PCR-RFLP, Sanger sequencing and new generation sequencing were used. In silico studies were performed with PolyPhen-2 software and the statistical analysis was done on SPSS software. The results obtained allowed to conclude that a low HbF level is a risk factor for stroke development (p=0.005). It was also observed that the patients with the rarest genotypes in HBG2 (rs7482144_TT+TC) present higher levels of HbF (p=0.031). It was concluded that, in BCL11A gene, the rs11886868_CC genotype and the rs4671393_allele_A seemed to predispose to higher levels of HbF. On the other hand, the polymorphisms studied in HBS1L-MYB did not reveal an association with HbF levels. The analysis of KLF1 gene revealed that 82.8% of the patients presented at least one variant in this gene. In this screening, there were detected 11 different variants, being one of them novel - the Q342H - whose effect at the protein level should be benign. We conclude that it is of great interest to consider the whole effect of the variants in the genes analyzed, since at the individual level, the combination of the rarest variants versus the more common is related with the HbF levels (p=0.021), constituting a genetic pattern with prognostic value. The genetic factors analyzed in this work have shown to be important modifiers of HbF levels and may become therapeutic targets.
Sickle Cell Anemia (SCA) is a hereditary hemolytic anemia with autosomal recessive transmission caused by the HBB:c.20A>T mutation in the β-globin gene, giving rise to hemoglobin S (HbS). Deoxygenated HbS polymerizes in the red cell, which acquires a sickle shape and becomes rigid and fragile. SCA is characterized by a variable clinical severity, including chronic hemolysis, vaso-occlusive episodes, and ultimately stroke. This phenotypic variability is due to environmental and genetic factors, and level of fetal hemoglobin (Hb F) has been proposed as the more important disease modulator. In this work, we aimed to investigate if the level of HbF in children with SCA is modulated by their genotypes in known polymorphic regions in the genes HBG2, BCL11A, HBS1L-MYB and KLF1. We also aimed to investigate whether the level of HbF in these children is related with the degree of hemolysis and the risk of stroke. We have studied 67 children with SCA, of African origin. These children were grouped in accordance with their degree of cerebral vasculopathy (Control, Risk and Stroke). For the molecular analysis, methodologies such as PCR-RFLP, Sanger sequencing and new generation sequencing were used. In silico studies were performed with PolyPhen-2 software and the statistical analysis was done on SPSS software. The results obtained allowed to conclude that a low HbF level is a risk factor for stroke development (p=0.005). It was also observed that the patients with the rarest genotypes in HBG2 (rs7482144_TT+TC) present higher levels of HbF (p=0.031). It was concluded that, in BCL11A gene, the rs11886868_CC genotype and the rs4671393_allele_A seemed to predispose to higher levels of HbF. On the other hand, the polymorphisms studied in HBS1L-MYB did not reveal an association with HbF levels. The analysis of KLF1 gene revealed that 82.8% of the patients presented at least one variant in this gene. In this screening, there were detected 11 different variants, being one of them novel - the Q342H - whose effect at the protein level should be benign. We conclude that it is of great interest to consider the whole effect of the variants in the genes analyzed, since at the individual level, the combination of the rarest variants versus the more common is related with the HbF levels (p=0.021), constituting a genetic pattern with prognostic value. The genetic factors analyzed in this work have shown to be important modifiers of HbF levels and may become therapeutic targets.
Description
Dissertação de mestrado em Análises Clínicas apresentada ao Instituto Superior de Ciências da Saúde Egas Moniz, 2017. Defendida em 14/7/217
Orientadora Doutora Paula Faustino (Instituto Nacional de Saúde Doutor Ricardo Jorge); Coorientadora Prof. Doutora Manuela Caniça (Instituto Superior de Ciências da Saúde Egas Moniz)
Trabalho desenvolvido no Instituto Nacional de Saúde Doutor Ricardo Jorge
Orientadora Doutora Paula Faustino (Instituto Nacional de Saúde Doutor Ricardo Jorge); Coorientadora Prof. Doutora Manuela Caniça (Instituto Superior de Ciências da Saúde Egas Moniz)
Trabalho desenvolvido no Instituto Nacional de Saúde Doutor Ricardo Jorge
Keywords
Hemoglobinopatias Doenças raras Doenças Genéticas globina hemoglobina drepanocitose Acidente Vascular Cerebral Anemia das células falciformes hemoglobina fetal fatores genéticos modificadores