A molecular tool to assess the pathological relevance of alpha-globin DNA variants
| Autor: | Christopher Newbound, Michelle Jennens, Talal Qadah, Reza Ghassemifar, Paula Holmes, Dianne Grey, John Beilby, Jill Finlayson, Nicole Pell |
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| Rok vydání: | 2012 |
| Předmět: |
Adult
Heterozygote In silico RNA Splicing DNA Mutational Analysis Biology medicine.disease_cause Pathology and Forensic Medicine alpha-Thalassemia medicine Humans Point Mutation Computer Simulation Cloning Molecular Pathology Molecular Gene Genetics Glycated Hemoglobin Mutation Expression vector Splice site mutation Reverse Transcriptase Polymerase Chain Reaction Point mutation Intron RNA splicing Female RNA Splice Sites |
| Zdroj: | Pathology. 44(4) |
| ISSN: | 1465-3931 |
| Popis: | Summary Aim While the phenotype for heterozygous beta-thalassae-mia 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 a-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. |
| Databáze: | OpenAIRE |
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