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Title: The development of molecular assays for the detection and identification of inherited disorders of haemoglobin in a highly heterogeneous population
Author: Turner, Andrew
ISNI:       0000 0004 7224 803X
Awarding Body: University of the West of England
Current Institution: University of the West of England, Bristol
Date of Award: 2018
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Inherited disorders of haemoglobin are the most common monogenic diseases in the world. These conditions arise as a result of deletions or point mutations affecting the globin genes. Gap polymerase chain reaction (Gap-PCR) is commonly used to detect globin gene deletions, however this technique is not ideal since it is time consuming and only able to detect known deletions with well-defined breakpoints. To address these issues an alternative technique called Gene Ratio Assay Copy Enumeration (GRACE) PCR was developed. In contrast to Gap-PCR, GRACE-PCR is a rapid, closed tube technique, requiring no further hands on time after the initial PCR setup. Furthermore, since it is based on copy number determination, GRACE-PCR does not require prior knowledge of the breakpoints and can thus be used for the detection of both known and novel deletions. GRACE-PCR methods were validated for the most clinically significant globin genes, HBA1, HBA2 and HBB. The large number of point mutations associated with the HBB gene make gene scanning by High Resolution Melting (HRM) PCR a potentially attractive diagnostic method. HRM-PCR assays have been previously described, however interference from certain Single Nucleotide Polymorphisms (SNPs) limited their ability to detect some mutations. Through the use of unlabelled probes and primers incorporating universal bases, it was possible to develop a more universally applicable HRM-PCR assay that was not affected by common SNPs. Additionally, a number of examples of two very rare haemoglobins, Hb Fontainebleau and Hb Handsworth were encountered during the course of this work. Each of these variants had previously been reported in just ten individuals worldwide. The diagnostic features of these two rare variants are described.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Thalassaemia ; haemoglobin ; Hb Fontainebleau ; Hb Handsworth ; GRACE-PCR