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)

Abstract

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.

Keywords: Beta thalassaemia; Copy number determination; GRACE-PCR; Gene quantification; HRM.

Fig. 1

Schematic representation the HBB gene showing amplicons and probe positions. A total of 11 primer pairs were used to scan the HBB gene by HRM-PCR (H1 to H11). In addition two primer pairs where used to test for deletions by GRACE-PCR (G1 and G2). The unlabelled probe (indicated by P) was used in conjunction with primer set H2. Primer sets H6 and H7 incorporated the universal base inosine in place of common non-pathological SNPs (See sequence information in Table 1)

Fig. 2

Representative HRM-PCR difference plots. Difference plots show wild type samples and the various mutants: 1a&b – genotype 37; 2a&b – genotype 28; 3a&b – genotype 33; 4a&b – genotype 2; 5a&b – genotypes 24 and 18 (see Table 3). Plots 1a&b were generated using primer pair 6 containing the universal base inosine (Table 1). Panels a and b show different runs with different samples

Fig. 3

Representative unlabelled probe HRM-PCR difference plots. Addition of the unlabeled probe allows differentiation of the pathological HBB:c.19G > A heterozygotes from common non-pathological HBB:c.9C > T heterozygotes; and of HBB:c.20A > T homozygotes from HBB:c.9C > T homozygotes. The unlabeled probe produced distinct probe and amplicon melt regions (a). Certain pathological variants cannot be distinguished from the HBB:c.9C > T SNP in the amplicon melt region (b), but are clearly separated in the probe melt region (c)

Fig. 4

GRACE-PCR to determine copy numbers of the HBB gene. Limited cycle GRACE-PCR was used to amplify targets from the CLCN7 reference gene [34] and the HBB gene. The raw melt curves (a) are difficult to interpret, but after normalization (b) the ratio of CLCN7:HBB gene copy numbers is easily visualized