title: A molecular tool to assess the pathological relevance of alpha-globin DNA variants.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Haematology, PathWest Laboratory Medicine, QEII Medical Centre, Nedlands, Western Australia, Australia.
authors.forename: Talal
authors.initials: T
authors.lastname: Qadah
authors.forename: Jill
authors.initials: J
authors.lastname: Finlayson
authors.forename: Christopher
authors.initials: C
authors.lastname: Newbound
authors.forename: Nicole
authors.initials: N
authors.lastname: Pell
authors.forename: Michelle
authors.initials: M
authors.lastname: Jennens
authors.forename: Paula
authors.initials: P
authors.lastname: Holmes
authors.forename: Dianne
authors.initials: D
authors.lastname: Grey
authors.forename: John
authors.initials: J
authors.lastname: Beilby
authors.forename: Reza
authors.initials: R
authors.lastname: Ghassemifar
mesh_terms: D000328:Adult; D003001:Cloning, Molecular; D003198:Computer Simulation; D004252:DNA Mutational Analysis; D005260:Female; D006442:Glycated Hemoglobin / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D057089:Pathology, Molecular / Q000379:methods*; D017354:Point Mutation; D022821:RNA Splice Sites / Q000235:genetics*; D012326:RNA Splicing; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017085:alpha-Thalassemia / Q000175:diagnosis* / Q000235:genetics
chemical_list: D006442:Glycated Hemoglobin; D022821:RNA Splice Sites