Abstract
ObjectivesInherited 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.MethodsThe 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.ResultsA 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 %.ConclusionWe 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.
Highlights
According to the World Health Organization (WHO), inherited disorders of haemoglobin are the most common monogenic disorders in the world
Amplicon and primer design When a common non-pathological Single nucleotide polymorphism (SNP) was present in a region that was optimal for primer placement, we substituted the nucleotide at the SNP position with the universal base inosine
Incorporating inosine into the primer allows equal amplification of both the wild-type and variant alleles [37]. This proved to be a good strategy for obtaining clear High Resolution Melting (HRM)-Polymerase chain reaction (PCR) profiles without interference by the non-pathological SNP
Summary
According to the World Health Organization (WHO), inherited disorders of haemoglobin are the most common monogenic disorders in the world. 868 mutations of the HBB gene have been described that have either a qualitative or quantitative effect on β-globin synthesis. These include point mutations, small insertions / deletions (indels) and larger. Qualitative defects are normally the result of point mutations and lead to the formation of abnormal β-globin chains, which in turn give rise to variant haemoglobins. Quantitative defects of the HBB gene either reduce, or totally stop, the synthesis of βglobin resulting in β-thalassaemia. Unlike α-thalassaemia, which is mostly caused by large deletions, the majority of β-thalassaemia alleles are the result of point mutations or small indels. It has been suggested that large deletions may account for as much as 10 % of β-thalassaemia mutations [4]
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