A molecular tool to assess the pathological relevance of alpha-globin DNA variants
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. Met...
| Main Authors: | , , , , , , , , |
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| Format: | Journal Article |
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2012
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| Online Access: | http://hdl.handle.net/20.500.11937/11254 |
| _version_ | 1848747756205637632 |
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| author | Qadah, T. Finlayson, J. Newbound, C. Pell, N. Jennens, M. Holmes, P. Grey, D. Beilby, J. Ghassemifar, Reza |
| author_facet | Qadah, T. Finlayson, J. Newbound, C. Pell, N. Jennens, M. Holmes, P. Grey, D. Beilby, J. Ghassemifar, Reza |
| author_sort | Qadah, T. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T06:54:12Z |
| format | Journal Article |
| id | curtin-20.500.11937-11254 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| last_indexed | 2025-11-14T06:54:12Z |
| publishDate | 2012 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-112542017-09-13T14:53:52Z A molecular tool to assess the pathological relevance of alpha-globin DNA variants Qadah, T. Finlayson, J. Newbound, C. Pell, N. Jennens, M. Holmes, P. Grey, D. Beilby, J. Ghassemifar, Reza 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. 2012 Journal Article http://hdl.handle.net/20.500.11937/11254 10.1097/PAT.0b013e328353a117 restricted |
| spellingShingle | Qadah, T. Finlayson, J. Newbound, C. Pell, N. Jennens, M. Holmes, P. Grey, D. Beilby, J. Ghassemifar, Reza A molecular tool to assess the pathological relevance of alpha-globin DNA variants |
| title | A molecular tool to assess the pathological relevance of alpha-globin DNA variants |
| title_full | A molecular tool to assess the pathological relevance of alpha-globin DNA variants |
| title_fullStr | A molecular tool to assess the pathological relevance of alpha-globin DNA variants |
| title_full_unstemmed | A molecular tool to assess the pathological relevance of alpha-globin DNA variants |
| title_short | A molecular tool to assess the pathological relevance of alpha-globin DNA variants |
| title_sort | molecular tool to assess the pathological relevance of alpha-globin dna variants |
| url | http://hdl.handle.net/20.500.11937/11254 |