Development and validation of a high throughput, closed tube method for the determination of haemoglobin alpha gene (HBA1 and HBA2) numbers by gene ratio assay copy enumeration-PCR (GRACE-PCR)

Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

Abstract

© 2015 Turner et al. Background: Deletions of the aα-globin genes are the most common genetic abnormalities in the world. Currently multiplex Gap-PCRs are frequently used to identify specific sets of common deletions. However, these assays require significant post-amplification hands on time and cannot be used to identify novel or unexpected deletions. The aim of the current study was to develop a rapid screening test for the detection of all deletions of the aα-globin genes that can be integrated into a high volume clinical laboratory workflow. Methods: A gene ratio assay copy enumeration (GRACE) PCR method was developed by simultaneous amplification of targets in the aα-globin genes (HBA1 and HBA2) and the chloride channel voltage sensitive 7 (CLCN7) reference gene. A novel application of High Resolution Melting (HRM) analysis then allowed rapid determination of aα-globin gene copy numbers. The assay was validated using 105 samples with previously determined and 62 samples with unknown aα-globin genotypes. Results: The GRACE-PCR assay detected abnormal aα-globin gene copy numbers in 108 of the 167 samples evaluated. The results were consistent with those from a commercial reverse hybridization assay and no allele drop out was observed. Conclusions: We have successfully developed and validated a GRACE-PCR screening test for the detection of deletions and duplications of the aα-globin genes. The assay is based on copy number determination and has the ability to detect both known and novel deletions of the aα-globin genes. It is a closed tube technique; consequently the risk of amplicon contamination is negligible. Amplification, detection and analysis can be completed within one hour, making it faster, cheaper and simpler than other existing tests and thus well suited as a rapid first step in a clinical laboratory workflow.

• Journal Article Type: Article
• Acceptance Date: Dec 4, 2015
• Online Publication Date: Dec 18, 2015
• Publication Date: Dec 18, 2015
• Deposit Date: Dec 10, 2015
• Publicly Available Date: Jan 10, 2019
• Journal: BMC Medical Genetics
• Electronic ISSN: 1471-2350
• Publisher: BioMed Central
• Peer Reviewed: Peer Reviewed
• Volume: 16
• Article Number: 115
• DOI: https://doi.org/10.1186/s12881-015-0258-y
• Keywords: alpha thalassemia, copy number determination, gene quantification, qPCR, GRACE-PCR
• Public URL: https://uwe-repository.worktribe.com/output/801943
• Publisher URL: http://dx.doi.org/10.1186/s12881-015-0258-y
• Contract Date: Jan 10, 2019