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Clinical application of a protocol based on universal next-generation sequencing for the diagnosis of beta-thalassaemia and sickle cell anaemia in preimplantation embryos
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.
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To control for allele overamplification, homozygous SNPs were changed to heterozygous if the fraction of reads supporting the reference allele was at least 6% of the total.
“To control for allele overamplification, homozygous SNPs were changed to heterozygous if the fraction of reads supporting the reference allele was at least 6% of the total (21).”
From Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos (2021) · cites this paper as [21]
The Discussion (p. 16) reports preferential amplification and says high sequencing depth can reveal under-represented alleles in heterozygous samples. The Data analysis section (p. 8) describes assessing read proportions, but neither passage states that homozygous SNP calls were changed to heterozygous at a 6% reference-read threshold. (LLM, conf=0.78)
Experimental — accuracy not yet measuredA step was implemented in the SNP calling pipeline to control for allele overamplification bias, changing homozygous calls to heterozygous if the fraction of reads supporting the reference allele is above the median value across samples.
“In addition, we implemented a step in our SNP calling pipeline to control for allele overamplification bias, which is a common issue with single cell amplified DNA (21).This step changes homozygous calls to heterozygous if the fraction of reads supporting the reference allele is above the median value across samples”
From Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos (2021) · cites this paper as [21]
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Cao, A., & Galanello, R. (2010) · Genetics in Medicine · doi:10.1097/gim.0b013e3181cd68ed · Cited by 722
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Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos
Alanis-Lobato, G., Zohren, J., McCarthy, A., Fogarty, N. M. E., Kubikova, N., Hardman, E., Greco, M., Wells, D., Turner, J. M. A., & Niakan, K. K. (2021) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2004832117 · Cited by 234
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Metadata
- Authors
- Kubikova, N., Babariya, D., Sarasa, J., Spath, K., Alfarawati, S., & Wells, D.
- Affiliations
- John Radcliffe Hospital (GB)
- Corresponding author
- Kubikova, Nada
- Venue
- Reproductive BioMedicine Online
- Publisher
- Elsevier BV
- Published
- 2018-08
- Volume
- 37
- Issue
- 2
- Pages
- 136-144
- ISSN
- 1472-6483 (ISSN-L)
- OpenAlex
- W2803716681
- Cited by
- 16
- References
- 1
- Citations by year
- 2025: 2 · 2023: 3 · 2022: 1 · 2021: 3 · 2020: 1 · 2019: 3 · 2018: 3
- FWCI
- 2.78
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Reproductive BioMedicine Online (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute for Health Research
- University of Oxford Clarendon Fund
- Topics
- Health Sciences › Medicine › Pediatrics, Perinatology and Child Health › Prenatal Screening and Diagnostics
- Health Sciences › Medicine › Genetics (clinical) › Hemoglobinopathies and Related Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
- Keywords
- preimplantation genetic testing, beta-thalassemia, sickle cell disease, beta-globin gene mutations, HBB, multiplex PCR, single nucleotide polymorphisms, trophectoderm biopsy, allele dropout, karyomapping, embryo genotyping
- MeSH terms
- Alleles, Anemia, Sickle Cell, Blastocyst, Female, Genetic Testing, Genotype, Humans, Mutation, Pregnancy, beta-Thalassemia, Preimplantation Diagnosis, High-Throughput Nucleotide Sequencing
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