Rapid prenatal diagnosis of common beta-thalassemia mutations in Southeast Asia using pyrosequencing.

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.