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This dissertation, Assessing Alpha Globin Gene Amplification and Its Modification of Beta Thalassemia Phenotype by Yim-yu, Ngai, 倪艷茹, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Thalassemia is the most common inherited disorder in the world. The high degree of homologous sequences on the α2 and α1 genes leads to unequal crossover and generation of copy number changes. Patients with interaction of α globin gene amplification and β thalassemia trait may present with a β thalassemia intermedia phenotype. Individuals co-inherited with α globin gene amplification and α globin gene deletion also have their globin phenotype modified. Detection of alpha globin gene amplification is therefore useful in the clinical setting. Southern blotting analysis and polymerase chain reaction (PCR)-based assays have been used for this purpose. In this study, the ability of MLPA to determine the type and order of alpha globin gene amplification in 47 clinical samples with and without co-existing alpha globin gene deletion and beta thalassemia mutations was assessed. Data obtained from MLPA were used in genotype-phenotype correlation. The technique was also applied to determine the population frequency of alpha globin gene amplification. A real time PCR was set up and assessed for its ability to distinguish alpha globin gene triplication from normal. Results showed that MLPA was able to detect alpha globin gene amplification in all 47 cases, irrespective of the presence or absence of other co-existing alpha and beta globin gene defects. It also provides information on the type and exact order of amplification in all cases. Seven subjects were found to have a higher order of amplification than heterozygous triplication -heterozygous quadruplication and homozygous triplication. MLPA was able to diagnose complex alpha globin gene rearrangement in HK variant without resorting to another specific PCR test, Southern blotting analysis or family study. The modifying effect of alpha globin gene amplification on co-existing beta thalassemia mutation was demonstrated, showing in general a worsening effect on the clinical phenotype depending on the predicted degree of alpha and beta globin chain imbalance. Importantly, a less than perfect prediction was observed in the group of compound heterozygous alpha triplication and beta-zero thalassemia mutation, where patients could present with an intermedia or trait phenotype. This indicated the presence of other undermined disease phenotype modifiers. The modifying effect of alpha amplification on co-existing alpha globin gene deletion was also apparent. Of note was that an alpha SEA deletion was not masked by heterozygous alpha triplication. In the population study, MLPA revealed a relatively low alpha globin gene amplification of 1.04%. Analysis of the real time PCR set up showed a significant difference in the mean Cp ratio value between the heterozygous alpha triplication group and normal group. However, a diagnostic cutoff could not be established. Further modification of the method was needed. MLPA is a useful technique for the detection of alpha globin gene amplification in the clinical setting. The alpha globin gene copy number change data obtained help in the genotype-phenotype correlation in complex cases with co-existing alpha globin gene deletion and/or beta thalassemia mutations and therefore facilitate genetic counseling. However, there are other disease modifiers which contribute to clinical heterogeneity in patients with the same degree of alpha globin gene amplification. Application MLPA in a po

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