Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR)

Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

Abstract

© 2016 The Author(s). Objectives: Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. Methods: The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. Results: A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. Conclusion: We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts.

Citation

Turner, A., Sasse, J., & Varadi, A. (2016). Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR). BMC Medical Genetics, 17, Article 75. https://doi.org/10.1186/s12881-016-0334-y

• Journal Article Type: Article
• Acceptance Date: Sep 29, 2016
• Online Publication Date: Oct 19, 2016
• Publication Date: Oct 19, 2016
• Deposit Date: Oct 25, 2016
• Publicly Available Date: Oct 25, 2016
• Journal: BMC Medical Genetics
• Electronic ISSN: 1471-2350
• Publisher: BioMed Central
• Peer Reviewed: Peer Reviewed
• Volume: 17
• Article Number: 75
• DOI: https://doi.org/10.1186/s12881-016-0334-y
• Keywords: Beta thalassaemia, Copy number determination, Gene quantification, HRM, GRACE-PCR
• Public URL: https://uwe-repository.worktribe.com/output/907140
• Publisher URL: http://dx.doi.org/10.1186/s12881-016-0334-y