Abstract
BackgroundDeletions 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.MethodsA 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.ResultsThe 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.ConclusionsWe 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 materialThe online version of this article (doi:10.1186/s12881-015-0258-y) contains supplementary material, which is available to authorized users.
Highlights
Deletions of the α-globin genes are the most common genetic abnormalities in the world
Deletion of three genes causes a form of thalassaemia intermedia known as Hb H disease and often results in significant anaemia
Samples This study was conducted using anonymous, archived material from blood specimens submitted to the Sheikh Khalifa Medical City (SKMC) laboratory, Abu Dhabi, United Arab Emirates for α-thalassaemia screening
Summary
Deletions of the α-globin genes are the most common genetic abnormalities in the world. Multiplex Gap-PCRs are frequently used to identify specific sets of common deletions. These assays require significant post-amplification hands on time and cannot be used to identify novel or unexpected deletions. The World Health Organization (WHO) estimates that 20 % of the world’s population may have a deletion in one or more α-globin genes [1]. Deletion of three genes causes a form of thalassaemia intermedia known as Hb H disease and often results in significant anaemia. It has been estimated that around 13,000 pregnancies are affected annually by severe forms of α-thalassaemia [1]
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