Clinical application of a protocol based on universal next-generation sequencing for the diagnosis of beta-thalassaemia and sickle cell anaemia in preimplantation embryos

Research question Mutations of the beta-globin gene (HBB) cause beta-thalassaemia and sickle cell anaemia and are the most common cause of severe inherited disease in humans. Traditional PGD protocols for the detection of HBB mutations frequently involve labour intensive, patient-specific test design due to the wide diversity of disease associated HBB mutations. For this reason, we aimed to develop and clinically apply a universal PGD method to test for mutations in the HBB gene. Design A multiplex PCR protocol was designed, allowing simultaneous amplification of multiple overlapping DNA fragments encompassing the entire HBB gene sequence in addition to 17 characterized, closely linked single nucleotide polymorphisms (SNPs). Amplicons were then analyzed using a next generation sequencing (NGS) method, revealing mutations and SNP genotypes. The protocol was extensively validated, optimized and eventually clinically applied on whole-genome amplified (WGA) DNA derived from embryos of three couples carrying different combinations of β-thalassemia mutations. Results The HBB mutation status and associated SNP haplotypes were successfully determined in all 21 embryos. Interestingly, analysis of 141 heterozygous sites showed no instances of allele dropout (ADO) and the test displayed 100% concordance when compared to the results obtained from karyomapping. This suggests that the combination of trophectoderm biopsy and highly sensitive NGS may provide superior accuracy than typically achieved using traditional PGD methods. Importantly, no patient-specific test design or optimization was needed. Conclusions It is hoped that protocols that deliver almost universally-applicable low-cost tests, without compromising diagnostic accuracy, will improve patient access to PGD, especially in less affluent parts of the world.