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)

Abstract

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.

Fig. 1

A schematic representation of the α-globin locus from the Haemoglobin Zeta (HBZ) to the Haemoglobin Theta 1 (HBQ1) gene. The approximate position of the gene deletions detected in the current study is shown. The location of amplicons targeting the HBA1 and HBA2 genes are indicated by the arrows A1 and A2, respectively. The CLCN7 gene is located approximately 1.3 million bases 3′ of the α-globin locus (not shown)

Fig. 2

GRACE-PCR assay, limited cycle multiplex PCR targeting both α-globin genes (HBA1 and HBA2) and a reference gene (CNCN7). Peak height, relative to the reference gene, in the melting plots (−dF/dT versus temperature) is proportional to the copy number of each gene (indicated in parenthesis under the gene name). Four genotypes with varying α-globin gene copy numbers are shown in plots a to d

Fig. 3

Normalized ratios of -dF/dT peak heights for different copy numbers of the HBA1 and HBA2 genes. The horizontal bars indicate the mean ratio and the vertical bars indicate the range from the minimum to maximum ratio observed for each gene copy number. The data was normalized with the equation: Normalized ratio = (R_{WT Control}/T_{WT Control})/(R_Sample/T_Sample), where R and T is reference gene and target gene peak heights, respectively

Fig. 4

Normalization of GRACE-PCR assay to determine gene copy numbers. Raw melting curves show three distinct steps corresponding to the melting of the CLCN7, HBA1 and HBA2 gene products generated during the GRACE-PCR Screening Test (a). Setting the normalization regions N1 and N2 (dark grey bars) in the HRM analysis software allowed the gene copy number ratio for CLCN7:HBA1 to be determined (b). Re-setting the normalization regions to N3 and N4 (light grey bars) allowed the determination of the gene copy number ratio HBA1:HBA2 (c). The copy number for CLCN7 is assumed to be two, which allows the number of HBA1 copies to be calculated, which in turn allows the determination of the number of HBA2 copies