Use of an automated pyrosequencing technique for confirmation of Sickle Cell Disease

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 S{beta}0 thalassemia from SS or identify other hemoglobin variants, and in this case, hemoglobin (HBB) gene sequencing may be necessary.\n\nObjectivesTo develop a high throughput DNA based confirmatory assay for SCD and to detect mutations in the HBB gene.\n\nMethodsWe 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.\n\nResultsWe identified 168 samples with discrepant results between HPLC and PyS and 100 with concordant HPLC and PyS= heterozygous S, which would suggest S{beta}-thalassemia or other hemoglobin 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 S{beta} thalassemia genotype, 84 samples were classified as S{beta}0 thalassemia and 81 as S{beta}+ thalassemia. The most frequent beta thalassemia mutations of S{beta}0 and S{beta}+ were HBB: c.118C>T (Gln40Stop) and HBB c.92 + 6T> C, respectively.\n\nDiscussionThe 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 {beta}+ and {beta}0 mutations in SCD patients with S{beta}-thalassemia in a large multi-institutional SCD cohort in Brazil.