title: Experimental Characterization of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) and Hb Boghé (HBA2: c.177 C > A, p.His > Gln) Reveals Contradictory HBA2 Expression and Translation Patterns Despite Identical Amino Acid Substitutions.
abstract: In this study, we describe the clinical features and provide experimental analyses of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) that contrasted with Hb Boghé (HBA2: c.177 C > A, p.His > Gln). Despite the identical amino acid substitution in both variants, Hb Flurlingen shows the phenotype of α-thalassemia (α-thal), whereas Hb Boghé has no impact on α2-globin (HBA2) production. For in vitro transcription analysis, HBA2 expression constructs carrying the HBA2-WT (wild type), Hb Flurlingen and Hb Boghé sequences were generated and expressed in human bladder carcinoma 5637 cells for downstream analyses by quantitative real time-polymerase chain reaction (qReTi-PCR) and immunofluorochemistry (IFC). In silico analysis of secondary folding structures of the HBA2-WT, Hb Flurlingen and Hb Boghé mRNA sequences was performed using Mfold software. The gene transcription and translation analyses revealed that cells transfected with the Hb Flurlingen construct had significantly lower HBA2 transcription (-55.4%, p ≤ 0.01) and reduced protein synthesis when compared to the wild type group. In contrast, cells transfected with the Hb Boghé construct showed no significant changes in HBA2 transcription or translation activities when compared to the wild type group. The in silico prediction of possible effects of these mutations on the folding structures of the HBA2 transcripts showed a change of secondary folding pattern in the Hb Flurlingen transcript when compared to those of HBA2-WT and Hb Boghé. Our experimental findings support the clinical presentation of an α-thalassemic phenotype for Hb Flurlingen in contrast with Hb Boghé, despite identical amino acid substitutions. The results confirm the importance of experimental analysis in establishing the impact of novel base substitutions.
authors.affiliation: a Department of Haematology, PathWest Laboratory Medicine , Queen Elizabeth II Medical Centre , Nedlands , Western Australia.
authors.forename: Talal
authors.initials: T
authors.lastname: Qadah
authors.forename: Jill
authors.initials: J
authors.lastname: Finlayson
authors.forename: Maxine
authors.initials: M
authors.lastname: Dennis
authors.forename: Christopher
authors.initials: C
authors.lastname: Newbound
authors.forename: Reza
authors.initials: R
authors.lastname: Ghassemifar
mesh_terms: D000293:Adolescent; D019943:Amino Acid Substitution; D003062:Codon; D004252:DNA Mutational Analysis; D004909:Erythrocyte Indices; D005786:Gene Expression Regulation; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D006443:Hemoglobin A2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006453:Hemoglobinopathies / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D006455:Hemoglobins, Abnormal / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D007501:Iron / Q000097:blood; D008297:Male; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014168:Transferrin / Q000378:metabolism
chemical_list: D003062:Codon; D006455:Hemoglobins, Abnormal; D012333:RNA, Messenger; D014168:Transferrin; C401962:hemoglobin Boghe; D006443:Hemoglobin A2; D007501:Iron
keywords: exonic point mutation; mRNA folding; quantitative real-time polymerase chain reaction (qReTi-PCR); synonymous amino acid substitution; α-Thalassemia (α-thal)