One-step amplification refractory mutation system-PCR/high-resolution melting curve assay for carrier detection of red blood cell membranopathy caused by common SPTB mutations

Abstract

Introduction: Hereditary pyropoikilocytosis (HPP) is the most common cause of non-thalassemic severe inherited hemolytic anemia in Thai population. Up to 90% of affected patients harbor biallelic mutations of SPTB Providence (SPTB c.6055T>C), SPTB Buffalo (SPTB c.6074T>G), and SPTB Chiang Mai (SPTB c.6224A>G). This study aimed to develop a simple assay for mass screening of the three common SPTB mutations and to study their carrier frequencies in a healthy Thai population. Methods: We combined multiplex amplification refractory mutation system-PCR (ARMS-PCR) and high-resolution melting (HRM) curve analysis to create a one-step single-tube assay. The primers were designed to generate products with different melting temperatures in the presence of 6055C, 6074G, and 6224G. Internal control primers were added for quality control. Residual samples from blood donors and healthy adolescents were collected and tested for the three common SPTB mutations using the newly developed assay. Results: Optimized multiplex ARMS-PCR/HRM curve assay yielded well-separated melt curves to detect the three SPTB mutations with 4-h turnaround time. The assay was validated in screening of 2261 non-repetitive blood donors and 89 adolescents, in which 10 (0.43%), 2 (0.09%), and 3 (0.13%) individuals were identified as carriers of SPTB Providence, SPTB Buffalo, and SPTB Chiang Mai, respectively. All mutated SPTB and 20 random wild-type samples were confirmed using Sanger sequencing with 100% accuracy. Conclusion: The novel ARMS-PCR/HRM curve assay is simple, accurate, and time-effective for mass screening of the common SPTB mutations. This can be employed to prevent HPP birth in a Thai population.