Successful Strategy of Pre-implantation Genetic Testing for Beta-Thalassemia (c.17A>T Mutation)-Hb E Disease Using Multiplex Fluorescent PCR and Mini-Sequencing

Objectives: Hemoglobin E disease, c.26G>A variant of beta-globin gene, is the most common hemoglobinopathy in Asia. Compound heterozygotes inheriting Hb disease and beta-thalassemia generate beta-thalassemia-Hb with severe anemia. This study aimed to develop a pre-implantation genetic testing for monogenic disorders (PGT-M) protocol beta–thalassemia (c.17A>T mutation)-Hb (c.26G>A mutation) using multiplex fluorescent polymerase chain reaction (PCR) mini-sequencing. Materials Methods: bthalw1 primers were used amplify gene fragment covering both mutations, i.e. beta– thalassemia (c.17A>T) disease. D21S11 microsatellite marker was included contamination detection. Novel mini-sequencing designed tested detection mutations. Results: Pre-clinical work up optimized PGT-M 20 single buccal cells heterozygous subject showed 100% amplification efficiency 0% allele drop out (ADO) rate primers. In clinical cycle, 15 embryos subjected biopsy. The results two normal, one beta-thalassemia, six affected five ambiguous results. Two normally diagnosed chosen transfer, singleton pregnancy obtained. A healthy baby boy resulted. Postnatal confirmed PGT Conclusions: protocols PCR developed described here. applied cycle gave rise successful consequently boy. Mini-sequencing proved be rapid, accurate cost-effective PGT-M.