Análise quantitativa e molecular de hemoglobina fetal em indivíduos da população brasileira
Carregando...
Data
2003-01-01
Autores
Zamaro, Paula J. A. [UNESP]
Hidalgo, Claudia A. [UNESP]
Bonini-Domingos, Claudia R. [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Associação Brasileira de Hematologia e Hemoterapia e daSociedade Brasileira de Transplante de Medula Óssea
Resumo
A hemoglobina fetal - Hb F, formada por duas cadeias gama e duas cadeias alfa, é característica do período fetal do desenvolvimento, tendo sua síntese diminuída no período pós-natal. em algumas alterações hereditárias, a Hb F permanece aumentada, como nas delta-beta talassemia, beta talassemia e persistência hereditária de Hb F (PHHF). A síntese da globina gama também pode ser estimulada por fatores externos como leucemias, transplantes de medula óssea, induções químicas, dentre outros. Através da observação de Hb F aumentada em doadores de sangue por procedimentos eletroforéticos objetivou-se avaliar a quantidade de Hb F em amostras de sangue de candidatos à doação, visando estabelecer seus limites de normalidade na população de São José do Rio Preto e região, por meio de desnaturação alcalina e cromatografia líquida de alta pressão (HPLC), comparar as metodologias aplicadas e, nos indivíduos com Hb F aumentada, realizar estudos moleculares para identificar as mutações que alteram a expressão dos genes gama. Foram analisadas 208 amostras de sangue, sendo 119 de candidatos à doação e 89 de indivíduos sem sintomas de anemia ou achados hematológicos e com Hb F aumentada como grupo comparativo. Das 119 amostras de candidatos à doação, 110 foram utilizadas para traçar o perfil de normalidade de Hb F, comparando-se as metodologias de desnaturação alcalina e HPLC, onde se obteve a média de 1,48% e de 0,6%, respectivamente. A análise estatística por regressão linear mostrou diferença significativa na comparação entre as duas metodologias aplicadas, sendo a HPLC mais precisa para a quantificação de Hb F. Foram observados nos testes de rastreamento de hemoglobinas anormais nestas 110 amostras de sangue: 16,4% de alfa talassemia, 0,9% com Hb F aumentada, 0,9% com beta talassemia e 0,9% com hemoglobina variante de cadeia delta. Os outros nove doadores de sangue apresentaram Hb F acima de 10% em eletroforese e observou-se média de 32,28% para desnaturação alcalina e de 26,4% para HPLC. As análises moleculares por PCR-ASO foram realizadas na tentativa de se identificar um defeito genético que pudesse explicar o aumento de Hb F, pelo rastreamento de 16 mutações que originam talassemias do tipo beta. Encontraram-se 5,3% de heterozigotos para mutação CD6-A e 1,75% para as mutações CD 39, IVS1:6, -87 e IVS2:654, todas em heterozigose. Os resultados encontrados neste estudo evidenciam a necessidade de melhor caracterização dos perfis de hemoglobina obtidos pelos métodos clássicos e a importância de sua caracterização molecular.
The fetal hemoglobin, Hb F, formed by two alpha and two gamma globin chains, is characteristic of the fetal development period and has diminished synthesis in the post-natal period. In some hereditary alterations Hb F remains elevated, as in delta-beta thalassemia, beta thalassemia and hereditary persistence of Hb F (HPFH). The synthesis of gamma globin can also be stimulated by external factors including leukemia, blood marrow transplantation and chemical induction, amongst others. Through the observation of elevated levels of Hb F in blood samples using electrophoretic procedures, we aimed at evaluating the amount of Hb F in blood donor samples in order to establish limits of normality in the population from São José do Rio Preto and region. Also using alkaline denaturation and high-pressure liquid chromatography (HPLC), we compared the applied methodologies and, in individuals with increased Hb F, we used molecular studies to identify mutations that modify the gamma globin gene expression. Blood samples from 119 donation candidates and, as a control group, 89 from individuals without symptoms of anemia or hematological findings and with increased Hb F, were analyzed. of these 119 samples from blood donation candidates, 110 were used to trace the profile of normal Hb F, comparing the applied measurement methodologies, giving an average of 1.48% for alkaline denaturation and 0.6%, for HPLC. Statistical analysis using linear regression showed a significant difference between the two applied methodologies. HPLC was the best method to measure Hb F. In the 110 blood samples that were evaluated in the screening tests for abnormal hemoglobins 16.4% presented with alpha thalassemia, 0.9% with increased Hb F, 0.9% with beta thalassemia and 0.9% with a hemoglobin variant of the delta globin chain. The other nine blood donors presented with Hb F levels above 10% in electrophoresis with an average of 32.28% observed in alkaline denaturation and 26.4% in HPLC. Molecular analysis by PCR-ASO was carried out, screening the 16 mutations that give rise to beta-type thalassemia, in an attempt to identify a genetic defect that could explain the increase of Hb F. We found 5.3% of heterozygotes with the CD 6-A mutation and 1.75% with the CD 39, IVS1:6, -87 and IVS2:654 mutations. The results in this study suggested a necessity of better characterization of the hemoglobin profiles obtained by the classic methods and the importance of its molecular characterization.
The fetal hemoglobin, Hb F, formed by two alpha and two gamma globin chains, is characteristic of the fetal development period and has diminished synthesis in the post-natal period. In some hereditary alterations Hb F remains elevated, as in delta-beta thalassemia, beta thalassemia and hereditary persistence of Hb F (HPFH). The synthesis of gamma globin can also be stimulated by external factors including leukemia, blood marrow transplantation and chemical induction, amongst others. Through the observation of elevated levels of Hb F in blood samples using electrophoretic procedures, we aimed at evaluating the amount of Hb F in blood donor samples in order to establish limits of normality in the population from São José do Rio Preto and region. Also using alkaline denaturation and high-pressure liquid chromatography (HPLC), we compared the applied methodologies and, in individuals with increased Hb F, we used molecular studies to identify mutations that modify the gamma globin gene expression. Blood samples from 119 donation candidates and, as a control group, 89 from individuals without symptoms of anemia or hematological findings and with increased Hb F, were analyzed. of these 119 samples from blood donation candidates, 110 were used to trace the profile of normal Hb F, comparing the applied measurement methodologies, giving an average of 1.48% for alkaline denaturation and 0.6%, for HPLC. Statistical analysis using linear regression showed a significant difference between the two applied methodologies. HPLC was the best method to measure Hb F. In the 110 blood samples that were evaluated in the screening tests for abnormal hemoglobins 16.4% presented with alpha thalassemia, 0.9% with increased Hb F, 0.9% with beta thalassemia and 0.9% with a hemoglobin variant of the delta globin chain. The other nine blood donors presented with Hb F levels above 10% in electrophoresis with an average of 32.28% observed in alkaline denaturation and 26.4% in HPLC. Molecular analysis by PCR-ASO was carried out, screening the 16 mutations that give rise to beta-type thalassemia, in an attempt to identify a genetic defect that could explain the increase of Hb F. We found 5.3% of heterozygotes with the CD 6-A mutation and 1.75% with the CD 39, IVS1:6, -87 and IVS2:654 mutations. The results in this study suggested a necessity of better characterization of the hemoglobin profiles obtained by the classic methods and the importance of its molecular characterization.
Descrição
Palavras-chave
Fetal hemoglobin, Abnormal hemoglobin, molecular diagnosis, Hemoglobina fetal, Hemoglobinas anormais, Diagnóstico molecular
Como citar
Revista Brasileira de Hematologia e Hemoterapia. Associação Brasileira de Hematologia e Hemoterapia e daSociedade Brasileira de Transplante de Medula Óssea, v. 25, n. 4, p. 223-229, 2003.