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. These are the most common cause of severe inherited disease in humans. Traditional preimplantation genetic testing protocols for detecting HBB mutations frequently involve labour intensive, patient-specific test designs owing to the wide diversity of disease-associated HBB mutations. We, therefore, asked the question whether a universally applicable preimplantation genetic testing method can be developed to test for HBB gene mutations. DESIGN: A multiplex polymerase chain reaction 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 (SNP). Amplicons were then analysed using a next-generation sequencing method, revealing mutations and SNP genotypes. The protocol was extensively validated, optimized and eventually clinically applied on whole-genome amplified DNA derived from embryos of three couples carrying different combinations of beta-thalassaemia mutations. RESULTS: The HBB mutation status and associated SNP haplotypes were successfully determined in all 21 embryos. Analysis of 141 heterozygous sites showed no instances of allele dropout and the test displayed 100% concordance compared with the results obtained from karyomapping. This suggests that the combination of trophectoderm biopsy and highly sensitive next-generation sequencing may provide superior accuracy than typically achieved using traditional preimplantation genetic testing 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 preimplantation genetic testing, especially in less affluent parts of the world.