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)

BACKGROUND: Deletions of the α-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 α-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 α-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 α-globin gene copy numbers. The assay was validated using 105 samples with previously determined and 62 samples with unknown α-globin genotypes. RESULTS: The GRACE-PCR assay detected abnormal α-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 α-globin genes. The assay is based on copy number determination and has the ability to detect both known and novel deletions of the α-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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12881-015-0258-y) contains supplementary material, which is available to authorized users.