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

Autoři: Camila Cruz de Martino aff001;  Cecilia Salete Alencar aff002;  Paula Loureiro aff003;  Anna Barbara de Freitas Carneiro-Proietti aff004;  Claudia de Alvarenga Máximo aff005;  Rosimere Afonso Mota aff006;  Daniela Oliveira Werneck Rodrigues aff007;  Nelson Gaburo Junior aff001;  Shannon Kelly aff008;  Ester Cerdeira Sabino aff001
Působiště autorů: Instituto de Medicina Tropical de São Paulo, Laboratório de Parasitologia, LIM 46, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, Brazil aff001;  Laboratório de Investigacao Medica, LIM 03, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, São Paulo, Brazil aff002;  Hemope, Recife, Pernambuco, Brazil aff003;  Hemominas, Belo Horizonte, Minas Gerais, Brazil aff004;  Hemorio, Rio de Janeiro, Rio de Janeiro, Brazil aff005;  Hemominas, Montes Claros, Minas Gerais, Brazil aff006;  Hemominas, Juiz de Fora, Minas Gerais, Brazil aff007;  Vitalant Research Institute, San Francisco, California, United States of America aff008;  UCSF Benioff Children’s Hospital Oakland, Oakland, California, United States of America aff009
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article



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.

Klíčová slova:

Beta-thalassemia – Brazil – Dideoxy DNA sequencing – Hemoglobin – High performance liquid chromatography – Polymerase chain reaction – Sickle cell disease – Thalassemia


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