Preimplantation genetic diagnosis for β-thalassaemia using sequencing of single cell PCR products to detect mutations and polymorphic loci

In order to carry out preimplantation genetic diagnosis (PGD) for β-thalassaemia, we have applied direct sequencing of single cell PCR products to detect mutations and polymorphic loci within the β-globin gene. Conventional duplex PCR was used to amplify two regions of the β-globin gene with an amplification efficiency of 79% for blastomeres. Sequencing data were obtained for 100% of amplified products, with 12% having confirmed allele drop-out (ADO). A double ADO event was observed at least twice, confirming the real risk of such an event during PGD. In one couple, the presence of a polymorphism linked to the female partner’s mutation enabled us to eliminate the risk of misdiagnosis due to double ADO without having to amplify both mutations within the same PCR product. We present here the data from eight clinical PGD cycles for three couples resulting in a singleton pregnancy and a twin pregnancy with all babies confirmed to be free from β-thalassaemia (major).