A molecular tool to assess the pathological relevance of alpha-globin DNA variants
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Aim: While the phenotype for heterozygous beta-thalassaemia is straightforward, it is more difficult to confirm a causative relationship for mutations in the alpha-globin genes. The aim of this study was to generate an in vitro system to evaluate the pathological relevance of α-globin mutations. Methods: The novel variant HBA1:c.301-3C>G was used as a model. In silico analysis predicted an aberrant acceptor splice site in the mutant sequence. Subsequent in vitro studies included generation of and transfection of an expression vector carrying the HBA1:c.301-3C>G mutation, RNA purification, reverse-transcription polymerase chain reaction (RT-PCR) and cDNA sequencing. Immunofluorochemistry (IFC) with antibodies specific to the N- and or C- terminal of the α-globin protein was used in protein detection. Results: In vitro molecular characterisation of this point mutation confirmed the preferential utilisation of a cryptic splice site at intron 2 of the pre-mRNA, resulting in a shift in the reading frame causing a premature termination codon (PTC) at codons 101/102 and generation of a truncated protein. Conclusion: We have described here a molecular tool to study mutations that affect α-globin pre-mRNA splicing and translation. We confirm in silico predictions of the consequences of the HBA1:c.301-3C>G mutation, proving aberrant RNA splicing and the production of a truncated α-globin protein.
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In vitro characterization of the a-thalassemia point mutation HBA2:c.95+1G>A [IVS-I-1(G>A) (a2)]Qadah, T.; Finlayson, J.; Ghassemifar, Reza (2012)The a-thalassemias are a group of disorders occurring as a result of decreased synthesis of a-globin chains, most commonly due to deletions of a-globin genes. Detection of a-thalassemia (a-thal) caused by point mutations ...
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