Bases moleculares e aspectos diagnósticos das Talassemias Alfa e Beta no Brasil
Data
2019-09-26
Autores
Lavelle, Nathalya Cristina 
Orientadores
Rizzatti, Edgar Gil
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: As talassemias são doenças caracterizadas por alterações no ritmo de síntese de uma ou mais cadeias de globinas, com consequente ausência ou redução do número de cadeias a serem formadas. A depender dos genes envolvidos, são denominadas alfa- ou beta-talassemias. Nas talassemias, o número de moléculas de hemoglobina por célula é limitado, causando a microcitose (diminuição do volume corpuscular médio (VCM) das hemácias). A microcitose é comum na prática clínica e suas principais causas são: anemia ferropriva, beta talassemia e anemia da doença crônica. Chamou-nos a atenção, na rotina do setor de Hematologia do Laboratório Central do Grupo Fleury, localizado na cidade de São Paulo, SP, o elevado número de casos com microcitose, concentração de Hb A2 e perfil de ferritina normais. Essa observação sugere que os métodos de triagem empregados no diagnóstico das alfatalassemias podem não estar refletindo sua real ocorrência. Adicionalmente, a grande quantidade de casos com elevação de Hb A2 e microcitose, sugerindo betatalassemia, foi digna de nota. No Brasil, alguns estudos envolvendo métodos moleculares para diagnóstico das talassemias foram realizados, entretanto, a maioria desses estudos limitou-se a regiões específicas do país. Objetivos: Investigar por métodos moleculares a ocorrência da alfa-talassemia em pacientes com microcitose sem causas óbvias; identificar a presença de mutações causadoras de betatalassemia nos pacientes com microcitose e elevação de Hb A2, e determinar a prevalência de ambas as condições em diferentes regiões do Brasil. Correlacionar os achados moleculares com o perfil hematológico da série vermelha e de hemoglobinas,das alfa- e beta-talassemias. Pacientes e Métodos: No período de 15 de fevereiro a 1 de junho de 2016, foram analisadas 4815 amostras do setor de Hematologia,provenientes dos estados: Bahia, Distrito Federal, Paraná, Pernambuco, Rio de Janeiro, Rio Grande do Sul e São Paulo. Excluiu-se 314 amostras devido à idade dos pacientes ser inferior a 2 anos. Nas 4501 amostras restantes, aplicou-se os critérios de inclusão para quadros sugestivos de alfa- ou beta-talassemias, respectivamente: combinação de microcitose e dosagem de Hb A2 normal (2,0% a 3,5%) ou diminuída (<2,0%), e dosagem de ferritina normal; ou presença de microcitose e dosagem de Hb A2 aumentada (>3,5%). Foram elegíveis 500 amostras consecutivas, sendo 208 sugestivas de beta-talassemia, e 292 sugestivas de alfa-talassemia. As técnicas de Gap-PCR Multiplex e Amplificação de Múltiplas Sondas Dependente de Ligação (MLPA) foram utilizadas para os casos com suspeita de alfa-talassemia, e o sequenciamento de DNA pelo método de Sanger para os casos sugestivos de betatalassemia. Resultados: Identificaram-se 194 casos (93,3%) positivos para betatalassemia, com 15 tipos de mutações, sendo as mais frequentes: CD 39 em 42,3% dos casos; 19,2% de IVS-I-6, e 17,3% da mutação IVS-I-110. Dentre elas, houve diferença nos índices de VCM e dosagem de Hb A2 (p<0,001). A alfa-talassemia foi confirmada em 180 casos (61,6%) submetidos ao teste de Gap-PCR Multiplex; 112 casos (38,4%) foram negativos. Dentre os casos positivos, 126 indivíduos apresentaram deleções em heterozigose (-α3.7, -α 4.2, e -α3.7/-α4.2), e outros 54, deleção -α 3.7 em homozigose. Os pacientes homozigotos apresentaram maior índice de microcitose (p<0,001) quando comparados aos heterozigotos. As amostras de indivíduos com resultado negativo no Gap-PCR, foram submetidas ao teste de MLPA. Dos 112 casos avaliados, encontrou-se 9 positivos (--MED, --HS-40, ααα anti3.7). Dentre os Estados avaliados, o Paraná apresentou maior frequência de alfa-talassemia, e o Rio Grande do Sul de beta-talassemia. Conclusões: A grande quantidade de indivíduos diagnosticados com alfa-talassemia neste estudo (64,7%), indica que essa condição é subdiagnosticada no Brasil, e ressalta a importância da análise molecular para investigar as causas de microcitose nos casos em que as hipóteses mais comuns tenham sido excluídas. A deleção -α3.7 representou a maior causa de alfa-talassemia (94,7%), tendo sido identificada na Bahia, Paraná, Pernambuco, Rio de Janeiro, Rio Grande do Sul e São Paulo. As diferenças no perfil hematológico e no perfil de hemoglobinas observadas nas mutações CD 39, IVS-I-6, IVS-I-110 causadoras de beta-talassemia, assim como nos casos de alfa-talassemia em heterozigose e homozigose, indicam que os genótipos influenciam o quadro clínico-laboratorial. Este estudo é pioneiro em investigar o perfil laboratorial e molecular das alfa- e betatalassemias nas diferentes regiões do Brasil.
Introduction: Thalassemias are diseases characterized by changes in the rhythm of synthesis of one or more chains of globins, with consequent absence or reduction of the number of chains to be formed. Depending on the genes involved, they are called alphaor betathalassemias. In thalassemias, the number of hemoglobin molecules per cell is limited, causing microcytosis (decrease in mean corpuscular volume (MCV) of red blood cells). Microcytosis is common in clinical practice and its main causes are: iron deficiency anemia, beta thalassemia and anemia of chronic disease. The high number of cases with microcytosis, normal A2 hemoglobin (Hb A2) concentration, and normal ferritin profile was noticed in the routine of the Hematology sector of the Fleury Group Central Laboratory, São Paulo, SP, Brazil. This observation suggests that the screening methods employed in the diagnosis of alphathalassemias may not be reflecting their actual occurrence. Additionally, the large number of cases with elevation of Hb A2 and microcytosis, suggesting betathalassemia, was noteworthy. In Brazil, some studies involving molecular methods for the diagnosis of thalassemias were performed; however, most of those studies were limited to specific regions of the country. Objectives: To investigate by molecular methods the occurrence of alphathalassemia in patients with microcytosis without obvious causes; to identify the presence of betathalassemia mutations in patients with microcytosis and Hb A2 elevation, and to determine the prevalence of both conditions in different regions of Brazil. Also, to correlate the molecular findings with the hematological profile of the red series and hemoglobins, alphaand betathalassemias. Patients and Methods: In the period from February 15 to June 1, 2016, 4815 samples from the Hematology sector were analyzed, from Bahia, Distrito Federal, Paraná, Pernambuco, Rio de Janeiro, Rio Grande do Sul and São Paulo. Three hundred and fourteen samples were excluded because the patient's age was less than 2 years old. In the remaining 4501 samples, the inclusion criteria were applied for suggestive alphaor betathalassemia, respectively: combination of microcytosis and normal (2.0% to 3.5%) or decreased (<2,0%) Hb A2 concentration, and normal ferritin dosage; or presence of microcytosis and increased Hb A2 concentration (>3.5%). Five hundred consecutive samples were eligible, 208 of which were suggestive of betathalassemia and 292 suggestive of alphathalassemia. Multiplex GapPCR and Multiplex Ligationdependent Probe Amplification (MLPA) techniques were used for cases with suggestive alphathalassemia, and DNA sequencing for cases with suggestive betathalassemia. Results: A total of 194 (93.3%) betathalassemia positive cases were identified, with 15 types of mutations, the most frequent being: CD 39 in 42.3% of the cases; 19.2% IVSI6, and 17.3% of the IVSI110 mutation. Among these, there were differences in VCM indexes and Hb A2 concentration (p<0.001). Alphathalassemia was confirmed in 180 cases (61.6%) submitted to the Multiplex GapPCR test; 112 (38.4%) were negative. Among the positive cases, 126 individuals had deletions in heterozygosity (α3.7,α4.2, and α3.7/ α4.2), and the other 54, deletion α3.7 in homozygosis. The homozygous patients had a higher rate of microcytosis (p<0.001) when compared to heterozygotes. Individuals with a negative GapPCR result were tested for MLPA. Of the 112 evaluated cases, 9 were positive (MED, HS40, ααα anti3.7). Among the evaluated States, Paraná presented a higher frequency of alphathalassemia, and Rio Grande do Sul of betathalassemia.Conclusions: The large number of individuals diagnosed with alphathalassemia in this study (64.7%) indicates that this condition is underdiagnosed in Brazil and stresses the importance of molecular analysis to investigate the causes of microcytosis in cases where the most common hypothesis have been excluded. The α3.7 deletion was the major cause of alphathalassemia (94.7%). It was identified in Bahia, Paraná, Pernambuco, Rio de Janeiro, Rio Grande do Sul and São Paulo. Differences in the hematological profile and hemoglobin profile observed in mutations for CD 39, IVSI6, IVSI110 causing betathalassemia,as well as in heterozygous and homozygous alphathalassemia cases, indicate that genotypes influence the clinical laboratory situation. This study is pioneer in investigating the molecular and laboratorial profile of alpha and betathalassemias in different regions of Brazil.
Introduction: Thalassemias are diseases characterized by changes in the rhythm of synthesis of one or more chains of globins, with consequent absence or reduction of the number of chains to be formed. Depending on the genes involved, they are called alphaor betathalassemias. In thalassemias, the number of hemoglobin molecules per cell is limited, causing microcytosis (decrease in mean corpuscular volume (MCV) of red blood cells). Microcytosis is common in clinical practice and its main causes are: iron deficiency anemia, beta thalassemia and anemia of chronic disease. The high number of cases with microcytosis, normal A2 hemoglobin (Hb A2) concentration, and normal ferritin profile was noticed in the routine of the Hematology sector of the Fleury Group Central Laboratory, São Paulo, SP, Brazil. This observation suggests that the screening methods employed in the diagnosis of alphathalassemias may not be reflecting their actual occurrence. Additionally, the large number of cases with elevation of Hb A2 and microcytosis, suggesting betathalassemia, was noteworthy. In Brazil, some studies involving molecular methods for the diagnosis of thalassemias were performed; however, most of those studies were limited to specific regions of the country. Objectives: To investigate by molecular methods the occurrence of alphathalassemia in patients with microcytosis without obvious causes; to identify the presence of betathalassemia mutations in patients with microcytosis and Hb A2 elevation, and to determine the prevalence of both conditions in different regions of Brazil. Also, to correlate the molecular findings with the hematological profile of the red series and hemoglobins, alphaand betathalassemias. Patients and Methods: In the period from February 15 to June 1, 2016, 4815 samples from the Hematology sector were analyzed, from Bahia, Distrito Federal, Paraná, Pernambuco, Rio de Janeiro, Rio Grande do Sul and São Paulo. Three hundred and fourteen samples were excluded because the patient's age was less than 2 years old. In the remaining 4501 samples, the inclusion criteria were applied for suggestive alphaor betathalassemia, respectively: combination of microcytosis and normal (2.0% to 3.5%) or decreased (<2,0%) Hb A2 concentration, and normal ferritin dosage; or presence of microcytosis and increased Hb A2 concentration (>3.5%). Five hundred consecutive samples were eligible, 208 of which were suggestive of betathalassemia and 292 suggestive of alphathalassemia. Multiplex GapPCR and Multiplex Ligationdependent Probe Amplification (MLPA) techniques were used for cases with suggestive alphathalassemia, and DNA sequencing for cases with suggestive betathalassemia. Results: A total of 194 (93.3%) betathalassemia positive cases were identified, with 15 types of mutations, the most frequent being: CD 39 in 42.3% of the cases; 19.2% IVSI6, and 17.3% of the IVSI110 mutation. Among these, there were differences in VCM indexes and Hb A2 concentration (p<0.001). Alphathalassemia was confirmed in 180 cases (61.6%) submitted to the Multiplex GapPCR test; 112 (38.4%) were negative. Among the positive cases, 126 individuals had deletions in heterozygosity (α3.7,α4.2, and α3.7/ α4.2), and the other 54, deletion α3.7 in homozygosis. The homozygous patients had a higher rate of microcytosis (p<0.001) when compared to heterozygotes. Individuals with a negative GapPCR result were tested for MLPA. Of the 112 evaluated cases, 9 were positive (MED, HS40, ααα anti3.7). Among the evaluated States, Paraná presented a higher frequency of alphathalassemia, and Rio Grande do Sul of betathalassemia.Conclusions: The large number of individuals diagnosed with alphathalassemia in this study (64.7%) indicates that this condition is underdiagnosed in Brazil and stresses the importance of molecular analysis to investigate the causes of microcytosis in cases where the most common hypothesis have been excluded. The α3.7 deletion was the major cause of alphathalassemia (94.7%). It was identified in Bahia, Paraná, Pernambuco, Rio de Janeiro, Rio Grande do Sul and São Paulo. Differences in the hematological profile and hemoglobin profile observed in mutations for CD 39, IVSI6, IVSI110 causing betathalassemia,as well as in heterozygous and homozygous alphathalassemia cases, indicate that genotypes influence the clinical laboratory situation. This study is pioneer in investigating the molecular and laboratorial profile of alpha and betathalassemias in different regions of Brazil.
Descrição
Citação
LAVELLE, Nathalya Cristina. Bases moleculares e aspectos diagnósticos das talassemias alfa e beta no Brasil. 2019. 101f. Dissertação (Mestrado em Hematologia e Oncologia) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2019.