Rapid prenatal diagnosis of common beta‐thalassemia mutations in Southeast Asia using pyrosequencing. (21st July 2013)

• Record Type: Journal Article
• Authors: Ho, Sherry Sze Yee; Huan, Pei Tee; Leow, Gek Har; Ching, Leng Kee; Chiu, Lily; Law, Hai Yang; Koay, Evelyn S. C.
• Is Part Of: Prenatal diagnosis. Volume 33:Number 11(2013:Nov.)
• Page Start: 1017
• Page End: 1022
• Publication Date: 2013-07-21
• Subjects: Prenatal diagnosis -- Periodicals; Fetus -- Diseases -- Diagnosis -- Periodicals; Electronic journals; 618.32075
• DOI: 10.1002/pd.4183
• Languages: English
• ISSNs: 0197-3851

Abstract

ABSTRACT: Objective: Current methods of prenatal diagnosis to detect beta‐thalassemia are Sanger sequencing and reverse dot blot. These methods are time‐consuming and can prolong assay turnaround time. We aim to develop a sensitive and rapid method to detect 27 beta‐thalassemia mutations using pyrosequencing. Method: Pyrosequencing primer pairs and sequencing primers were designed to detect 27 most common beta‐thalassemia mutations found in Singapore. Pyrosequencing was performed on 191 DNA samples with known beta‐thalassemia mutations isolated from 143 peripheral blood and 48 prenatal samples (seven chorionic villus biopsies, 26 cultured amniocytes, 15 uncultured amniocytes). All mutations were validated with Sanger sequencing. Results: Pyrosequencing identified 210 alleles with beta‐thalassemia mutations and 82 alleles without mutations with 100% sensitivity (lower 95% confidence interval [CI], 97.8%) and 100% specificity (lower 95% CI, 94.4%). All pyrosequences were concordant with Sanger‐based sequences. Pyrosequencing was able to detect DNA concentrations as low as 2 ng, obviating the need for cell culture in volume‐restricted samples. Sample receipt‐to‐report assay turnaround times were 16 to 18 h (Sanger sequencing) and 4 to 6 h (pyrosequencing). Conclusion: Pyrosequencing is a rapid and sensitive method to detect common beta‐thalassemia mutations without the need for cell culture, thus reducing the assay turnaround time. © 2013 John Wiley & Sons, Ltd. Abstract : What's already known about this topic? The most common prenatal diagnostic methods to detect beta‐thalassemia mutations are Sanger sequencing and reverse dot blot. Large amounts of DNA are required, and AF culture is usually necessary. This prolongs the assay turnaround time. What does this study add? This study describes the utilization of pyrosequencing to detect beta‐thalassemia mutations in AF samples without cell culture. This reduces the assay turnaround time.