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

Nada Kubikova; Dhruti Babariya; Jonas Sarasa; Katharina Spath; Samer Alfarawati; Dagan Wells

doi:10.1016/j.rbmo.2018.05.005

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

Reprod Biomed Online. 2018 Aug;37(2):136-144. doi: 10.1016/j.rbmo.2018.05.005. Epub 2018 May 21.

Authors

Nada Kubikova¹, Dhruti Babariya², Jonas Sarasa³, Katharina Spath², Samer Alfarawati², Dagan Wells⁴

Affiliations

¹ University of Oxford, Nuffield Department of Women's and Reproductive Health, John Radcliffe Hospital, Level 3, Women's Centre, Oxford, OX3 9DU, UK. Electronic address: nada.kubikova@bnc.ox.ac.uk.
² CooperGenomics, Institute of Reproductive Sciences, Oxford Business Park North, Alec Issigonis Way, Oxford OX4 2HW, UK.
³ iGLS, C/Britania 7, Alicante, 03540, Spain.
⁴ University of Oxford, Nuffield Department of Women's and Reproductive Health, John Radcliffe Hospital, Level 3, Women's Centre, Oxford, OX3 9DU, UK.

PMID: 29853423
DOI: 10.1016/j.rbmo.2018.05.005

Abstract

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.

Keywords: Beta-thalassaemia; Next generation sequencing; Preimplantation genetic diagnosis; Sickle-cell anaemia.

MeSH terms

Alleles
Anemia, Sickle Cell / diagnosis*
Anemia, Sickle Cell / genetics
Blastocyst*
Female
Genetic Testing / methods*
Genotype
High-Throughput Nucleotide Sequencing*
Humans
Mutation
Pregnancy
Preimplantation Diagnosis / methods*
beta-Thalassemia / diagnosis*
beta-Thalassemia / genetics