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

T. Qadah, Jill Finlayson, M. Dennis, C. Newbound, Reza Ghassemifar

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    Abstract

    © 2015 Informa Healthcare USA, Inc. All rights reserved. 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.
    Original languageEnglish
    Pages (from-to)340-345
    JournalHemoglobin
    Volume39
    Issue number5
    DOIs
    Publication statusPublished - 2015

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    Amino Acid Substitution
    Transcription
    Substitution reactions
    Amino Acids
    Computer Simulation
    Phenotype
    Thalassemia
    Globins
    Real-Time Polymerase Chain Reaction
    Polymerase chain reaction
    Urinary Bladder
    Software
    Carcinoma
    Delivery of Health Care
    Messenger RNA
    Mutation
    Genes
    Proteins

    Cite this

    @article{ac6b375d70364d9f86b1331016b28d45,
    title = "Experimental Characterization of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) and Hb Bogh{\'e} (HBA2: c.177 C > A, p.His > Gln) Reveals Contradictory HBA2 Expression and Translation Patterns Despite Identical Amino Acid Substitutions",
    abstract = "{\circledC} 2015 Informa Healthcare USA, Inc. All rights reserved. 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 Bogh{\'e} (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 Bogh{\'e} 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 Bogh{\'e} 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 Bogh{\'e} 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 Bogh{\'e} 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 Bogh{\'e}. Our experimental findings support the clinical presentation of an α-thalassemic phenotype for Hb Flurlingen in contrast with Hb Bogh{\'e}, despite identical amino acid substitutions. The results confirm the importance of experimental analysis in establishing the impact of novel base substitutions.",
    author = "T. Qadah and Jill Finlayson and M. Dennis and C. Newbound and Reza Ghassemifar",
    year = "2015",
    doi = "10.3109/03630269.2015.1062393",
    language = "English",
    volume = "39",
    pages = "340--345",
    journal = "Hemoglobin",
    issn = "0363-0269",
    publisher = "Informa Healthcare USA",
    number = "5",

    }

    TY - JOUR

    T1 - 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

    AU - Qadah, T.

    AU - Finlayson, Jill

    AU - Dennis, M.

    AU - Newbound, C.

    AU - Ghassemifar, Reza

    PY - 2015

    Y1 - 2015

    N2 - © 2015 Informa Healthcare USA, Inc. All rights reserved. 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.

    AB - © 2015 Informa Healthcare USA, Inc. All rights reserved. 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.

    U2 - 10.3109/03630269.2015.1062393

    DO - 10.3109/03630269.2015.1062393

    M3 - Article

    VL - 39

    SP - 340

    EP - 345

    JO - Hemoglobin

    JF - Hemoglobin

    SN - 0363-0269

    IS - 5

    ER -