Use of an automated pyrosequencing technique for confirmation of sickle cell disease

• Published in: PloS one 2019-12, Vol.14 (12), p.e0216020
• Main Authors: de Martino, Camila Cruz, Alencar, Cecilia Salete, Loureiro, Paula, Carneiro-Proietti, Anna Barbara de Freitas, Máximo, Claudia de Alvarenga, Mota, Rosimere Afonso, Rodrigues, Daniela Oliveira Werneck, Gaburo Junior, Nelson, Kelly, Shannon, Sabino, Ester Cerdeira
• Format: Article
• Language: English
• Subjects: Acetates; Acetic acid; Anemia, Sickle Cell - diagnosis; Anemia, Sickle Cell - epidemiology; Anemia, Sickle Cell - genetics; Assaying; beta-Thalassemia - diagnosis; beta-Thalassemia - epidemiology; beta-Thalassemia - genetics; Biology and Life Sciences; Blood; Brazil - epidemiology; Cellulose; Cellulose acetate; Cellulose esters; Chromatography; Citric acid; Cohort Studies; Deoxyribonucleic acid; DNA; DNA sequencing; EDTA; Electrophoresis; Epidemiology; Gene sequencing; Genes; Genotype; Genotype & phenotype; Genotypes; Glycosylated hemoglobin; HBB gene; Hemoglobin; Hemoglobin, Sickle - genetics; Hemoglobins; High performance liquid chromatography; High-Throughput Nucleotide Sequencing - methods; Humans; Isoelectric focusing; Laboratories; Liquid chromatography; Medical screening; Medicine and Health Sciences; Methods; Mutation; Newborn infants; People and places; Polysaccharides; Research and Analysis Methods; Sickle cell anemia; Sickle cell disease; Sickle hemoglobin; Technology; Technology application; Thalassemia
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0216020

The 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. To develop a high throughput DNA based confirmatory assay for SCD and to detect mutations in the HBB gene. We 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. We 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. The 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.