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

Abstract

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

Diagnostic Accuracy of HemotypeSC as a Point-of-Care Testing Device for Sickle Cell Disease: Findings from a Southwestern State in Nigeria and Implications for Patient Care in Resource-Poor Settings of sub-Saharan Africa

“…We considered the latter the gold standard for SCD diagnosis and determination of other Hb genotypes. 15 We hypothesized that the performance of HemoTypeSC relative to the gold standard, is not significantly different from that of the 2 other methods. Findings from this study will further establish the accuracy and usefulness or otherwise of HemoTypeSC™ in diagnosing SCD and other Hb genotypes in limited-resource settings like SSA.…”

Genome-wide association study of ischemic stroke risk in Sickle Cell confirms ADAMTS2, CDK18, uncovers 12 novel loci

“…Whole blood was collected in EDTA, and DNA was extracted from the buffy coat via alcohol precipitation, quantified with rtPCR, and normalized to 10 ng/uL. SCD genotypes were confirmed using allele-specific pyrosequencing (Qiagen, Hilden, Germany) 23 and Sanger sequencing of exons 1 and 2 of HBB if the pyrosequencing results conflicted with medical records. Additional sequencing of HBB exon 3, introns, and promoter was performed for samples with unresolved genotypes at the Hemoglobinopathy Reference Laboratory at UCSF Benioff Children’s Hospital Oakland.…”