BackgroundThe diagnosis of sickle cell disease (SCD) is made by hemoglobin assays such as high-performance liquid chromatography (HPLC), isoelectric focusing and cellulose acetate or citrate agar electrophoresis. These assays are easy to perform and used in large-scale newborn screening in many countries. These tests however may not easily differentiate Sβ0 thalassemia from SS or identify other hemoglobin variants, and in this case, hemoglobin (HBB) gene sequencing may be necessary.ObjectivesTo develop a high throughput DNA based confirmatory assay for SCD and to detect mutations in the HBB geneMethodsWe developed an automated pyrosequencing technique (PyS) based on QIAGEN technology (Hilden, Germany) to detect homozygous or heterozygous hemoglobin S mutations as well as hemoglobin C mutations. The technique was tested on 2,748 samples from patients enrolled in a multi-center SCD cohort in Brazil. Patients were previously tested using HPLC to diagnose SCD as part of routine clinical care. Any subjects with discrepant results between HPLC and PyS or with heterozygous hemoglobin S detected had Sanger sequencing of the HBB gene.ResultsWe identified 168 samples with discrepant results between HPLC and PyS and 100 with concordant PyS = heterozygous S and HPLC, which would suggest SB-thalassemia or other heterozygous S variants. The PyS assay correctly identified 1906 (98.7%) of the 1930 HbSS and 628 (98.7%) of the 636 HbSC samples. Of the 179 remaining samples, PyS correctly indicated S heterozygosis in 165 (92.2%). Of the 165 heterozygous S samples confirmed by Sanger as consistent with Sβ thalassemia genotype, 84 samples were classified as Sβ0 thalassemia and 81 as Sβ+ thalassemia. The most frequent beta thalassemia mutations of Sβ0 and Sβ+ were HBB: c.118C>T (Gln40Stop) and HBB c.92 + 6T> C, respectively.DiscussionThe PyS proved to be satisfactory for large-scale confirmatory testing of hemoglobin mutation. Moreover, with this study we were able to describe the most common β+ and β0 mutations in SCD patients with Sβ-thalassemia in a large multi-institutional SCD cohort in Brazil.
22Background: The diagnosis of sickle cell disease (SCD) is made by hemoglobin assays such 23 as high-performance liquid chromatography (HPLC), isoelectric focusing and cellulose 24 acetate or citrate agar electrophoresis. These assays are easy to perform and used in large-25 scale newborn screening in many countries. These tests however may not easily differentiate 26 Sβ 0 thalassemia from SS or identify other hemoglobin variants, and in this case, hemoglobin 27 (HBB) gene sequencing may be necessary. Objectives: To develop a high throughput DNA 28 based confirmatory assay for SCD and to detect mutations in the HBB gene. Methods: We 29 developed an automated pyrosequencing technique (PyS) based on QIAGEN technology 30 (Hilden, Germany) to detect homozygous or heterozygous hemoglobin S mutations as well as 31 hemoglobin C mutations. The technique was tested on 2,748 samples from patients enrolled in 32 a multi-center SCD cohort in Brazil. Patients were previously tested using HPLC to diagnose 33 SCD as part of routine clinical care. Any subjects with discrepant results between HPLC and 34 PyS or with heterozygous hemoglobin S detected had Sanger sequencing of the HBB gene.35 Results: We identified 168 samples with discrepant results between HPLC and PyS and 100 36 with concordant HPLC and PyS= heterozygous S, which would suggest Sβ-thalassemia or 37 other hemoglobin S variants. The PyS assay correctly identified 1906 (98.7%) of the 1930 38 HbSS and 628 (98.7%) of the 636 HbSC samples. Of the 179 remaining samples, PyS 39 correctly indicated S heterozygosis in 165 (92.2%). Of the 165 heterozygous S samples 40 confirmed by Sanger as consistent with Sβ thalassemia genotype, 84 samples were classified 41 as Sβ 0 thalassemia and 81 as Sβ + thalassemia. The most frequent beta thalassemia mutations 42 of Sβ 0 and Sβ + were HBB: c.118C>T (Gln40Stop) and HBB c.92 + 6T> C, respectively.43Discussion: The PyS proved to be satisfactory for large-scale confirmatory testing of 44 hemoglobin mutation. Moreover, with this study we were able to describe the most common 45 3 β + and β 0 mutations in SCD patients with Sβ-thalassemia in a large multi-institutional SCD 46 cohort in Brazil. 47
RESUMOMartino CC. Prevalência de beta-talassemias em pacientes com anemia falciforme em quatro estados do Brasil [dissertação]. São Paulo: Faculdade de Medicina, Universidade de São Paulo; 2020.O diagnóstico da doença falciforme (DF) é feito por ensaios de hemoglobina (Hb), como cromatografia líquida de alta eficiência (HPLC), foco isoelétrico e eletroforese em acetato de celulose ou ágar citrato. Esses ensaios são de fácil execução e utilizados em exames de recém-nascidos em larga escala em muitos países. No entanto, esses testes podem não diferenciar facilmente a talassemia Sβ0 da SS ou identificar outras variantes da hemoglobina e, nesse caso, o sequenciamento genético do gene da beta globina (HBB) pode ser necessário. Com o objetivo de desenvolver um ensaio confirmatório com base no ácido desoxirribonucleico (DNA) de alta produtividade para DF e detectar mutações no gene HBB. Métodos: Desenvolveu-se uma técnica de pyrosequencimento automático (Pyro) de acordo com a tecnologia Qiagen (Hilden, Alemanha) para detectar mutações homozigotas ou heterozigotas da hemoglobina S (HbS), bem como mutações na hemoglobina C (HbC). A técnica foi testada em 2.748 amostras de pacientes inscritos em uma coorte multicêntrica da DF no Brasil. Os pacientes foram previamente testados usando HPLC para diagnosticar DF como parte dos cuidados clínicos de rotina. Quaisquer indivíduos com resultados discrepantes entre HPLC e Pyro ou com hemoglobina S (HbS) heterozigótica detectada realizaram sequenciamento de Sanger do gene HBB. Resultados: Foram identificadas 168 amostras com resultados discrepantes entre HPLC e Pyro e 100 com Pyro concordante = S heterozigótico e HPLC, o que sugeriria talassemia Sβ ou outras variantes S heterozigóticas. O ensaio Pyro identificou corretamente 1.906 (98,7%) das HbSS de 1.930 amostras e 628 (98,7%) das 636 amostras de Hemoglobinopatia SC (HbSC). Das 179 amostras restantes, o Pyro indicou corretamente heterozigose S em 165 (92,2%). Das 165 amostras heterozigotas de S confirmadas por Sanger como consistentes com o genótipo de talassemia Sβ, 84 amostras foram classificadas como talassemia Sβ0 e 81 como talassemia Sβ+. As mutações beta talassêmicas mais frequentes de Sβ0 e Sβ+ foram HBB:c.118C>T (Gln40Stop) e HBB:c.92+6T>C, respectivamente. Discussão: O Pyro provou ser satisfatório para testes confirmatórios em larga escala da mutação da hemoglobina. Além disso, este estudo possibilitou a descrição das mutações β+ e β0 mais comuns em pacientes com DF com Stalassemia em uma grande coorte multi-institucional de DF no Brasil.
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