Molecular and cellular analysis of three novel alpha2-globin gene promoter mutations [HBA2:c.-59C>T], [HBA2:c.-81C>A] and [HBA2:c.-91G>A] reveal varying patterns of transcriptional and translational activities

T. Qadah, Jill Finlayson, M.A. Dennis, Reza Ghassemifar

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    Abstract

    While point mutations affecting the promoter region of b-globin gene are widely described, there are no well characterised reports of any point mutations currently found in the promoter of the a2-globin (HBA2) gene. We present clinical and experimental data for three novel HBA2 gene core and proximal promoter mutations. Using an in vitro system designed to assess the impact of point mutations, the three novel [HBA2:c.-59C>T], [HBA2:c.-81C>A] and [HBA2:c.-91G>A] promoter mutations identified in three unrelated patients were analysed for HBA2 gene transcriptional and translational activities. Following the generation and transfection of expression vectors carrying each mutation, the HBA2 transcription activity of the promoters from each mutant was analysed with quantitative real time-PCR (qReTi-PCR) technique. Immunofluorochemistry (IFC) was used to analyse HBA2 protein synthesis. The analyses showed that [HBA2:c.59C>T] and [HBA2:c.-91G>A] mutant constructs caused significant reduction in the HBA2 transcription levels by 53.7% (p=0.0008) and 36.2% (p=0.004), respectively, resulting in markedly lower HBA2 protein labelling when compared to the wild type as shown with subsequent IFC analysis. Conversely, the [HBA2:c.-81C>A] construct showed no significant changes in either transcription (p=0.089) or in protein labelling when compared to the wild type. The equal pAmp transcription levels found in each group confirmed that the observed labelling differences were not due to varying transfection efficiencies. This study emphasises the importance of in vitro studies to establish the impact of base substitutions on the level of gene expression, and the value of these studies in clinicopathological correlation so that appropriate advice can be given in genetic counselling. © 2013 Royal College of Pathologists of Australasia.
    Original languageEnglish
    Pages (from-to)46-52
    JournalPathology
    Volume46
    Issue number1
    DOIs
    Publication statusPublished - 2014

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    Globins
    Point Mutation
    Mutation
    Genes
    Transfection
    Australasia
    Proteins
    Genetic Counseling
    Genetic Promoter Regions
    Real-Time Polymerase Chain Reaction
    Gene Expression
    In Vitro Techniques

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    title = "Molecular and cellular analysis of three novel alpha2-globin gene promoter mutations [HBA2:c.-59C>T], [HBA2:c.-81C>A] and [HBA2:c.-91G>A] reveal varying patterns of transcriptional and translational activities",
    abstract = "While point mutations affecting the promoter region of b-globin gene are widely described, there are no well characterised reports of any point mutations currently found in the promoter of the a2-globin (HBA2) gene. We present clinical and experimental data for three novel HBA2 gene core and proximal promoter mutations. Using an in vitro system designed to assess the impact of point mutations, the three novel [HBA2:c.-59C>T], [HBA2:c.-81C>A] and [HBA2:c.-91G>A] promoter mutations identified in three unrelated patients were analysed for HBA2 gene transcriptional and translational activities. Following the generation and transfection of expression vectors carrying each mutation, the HBA2 transcription activity of the promoters from each mutant was analysed with quantitative real time-PCR (qReTi-PCR) technique. Immunofluorochemistry (IFC) was used to analyse HBA2 protein synthesis. The analyses showed that [HBA2:c.59C>T] and [HBA2:c.-91G>A] mutant constructs caused significant reduction in the HBA2 transcription levels by 53.7{\%} (p=0.0008) and 36.2{\%} (p=0.004), respectively, resulting in markedly lower HBA2 protein labelling when compared to the wild type as shown with subsequent IFC analysis. Conversely, the [HBA2:c.-81C>A] construct showed no significant changes in either transcription (p=0.089) or in protein labelling when compared to the wild type. The equal pAmp transcription levels found in each group confirmed that the observed labelling differences were not due to varying transfection efficiencies. This study emphasises the importance of in vitro studies to establish the impact of base substitutions on the level of gene expression, and the value of these studies in clinicopathological correlation so that appropriate advice can be given in genetic counselling. {\circledC} 2013 Royal College of Pathologists of Australasia.",
    author = "T. Qadah and Jill Finlayson and M.A. Dennis and Reza Ghassemifar",
    year = "2014",
    doi = "10.1097/PAT.0000000000000023",
    language = "English",
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    pages = "46--52",
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    TY - JOUR

    T1 - Molecular and cellular analysis of three novel alpha2-globin gene promoter mutations [HBA2:c.-59C>T], [HBA2:c.-81C>A] and [HBA2:c.-91G>A] reveal varying patterns of transcriptional and translational activities

    AU - Qadah, T.

    AU - Finlayson, Jill

    AU - Dennis, M.A.

    AU - Ghassemifar, Reza

    PY - 2014

    Y1 - 2014

    N2 - While point mutations affecting the promoter region of b-globin gene are widely described, there are no well characterised reports of any point mutations currently found in the promoter of the a2-globin (HBA2) gene. We present clinical and experimental data for three novel HBA2 gene core and proximal promoter mutations. Using an in vitro system designed to assess the impact of point mutations, the three novel [HBA2:c.-59C>T], [HBA2:c.-81C>A] and [HBA2:c.-91G>A] promoter mutations identified in three unrelated patients were analysed for HBA2 gene transcriptional and translational activities. Following the generation and transfection of expression vectors carrying each mutation, the HBA2 transcription activity of the promoters from each mutant was analysed with quantitative real time-PCR (qReTi-PCR) technique. Immunofluorochemistry (IFC) was used to analyse HBA2 protein synthesis. The analyses showed that [HBA2:c.59C>T] and [HBA2:c.-91G>A] mutant constructs caused significant reduction in the HBA2 transcription levels by 53.7% (p=0.0008) and 36.2% (p=0.004), respectively, resulting in markedly lower HBA2 protein labelling when compared to the wild type as shown with subsequent IFC analysis. Conversely, the [HBA2:c.-81C>A] construct showed no significant changes in either transcription (p=0.089) or in protein labelling when compared to the wild type. The equal pAmp transcription levels found in each group confirmed that the observed labelling differences were not due to varying transfection efficiencies. This study emphasises the importance of in vitro studies to establish the impact of base substitutions on the level of gene expression, and the value of these studies in clinicopathological correlation so that appropriate advice can be given in genetic counselling. © 2013 Royal College of Pathologists of Australasia.

    AB - While point mutations affecting the promoter region of b-globin gene are widely described, there are no well characterised reports of any point mutations currently found in the promoter of the a2-globin (HBA2) gene. We present clinical and experimental data for three novel HBA2 gene core and proximal promoter mutations. Using an in vitro system designed to assess the impact of point mutations, the three novel [HBA2:c.-59C>T], [HBA2:c.-81C>A] and [HBA2:c.-91G>A] promoter mutations identified in three unrelated patients were analysed for HBA2 gene transcriptional and translational activities. Following the generation and transfection of expression vectors carrying each mutation, the HBA2 transcription activity of the promoters from each mutant was analysed with quantitative real time-PCR (qReTi-PCR) technique. Immunofluorochemistry (IFC) was used to analyse HBA2 protein synthesis. The analyses showed that [HBA2:c.59C>T] and [HBA2:c.-91G>A] mutant constructs caused significant reduction in the HBA2 transcription levels by 53.7% (p=0.0008) and 36.2% (p=0.004), respectively, resulting in markedly lower HBA2 protein labelling when compared to the wild type as shown with subsequent IFC analysis. Conversely, the [HBA2:c.-81C>A] construct showed no significant changes in either transcription (p=0.089) or in protein labelling when compared to the wild type. The equal pAmp transcription levels found in each group confirmed that the observed labelling differences were not due to varying transfection efficiencies. This study emphasises the importance of in vitro studies to establish the impact of base substitutions on the level of gene expression, and the value of these studies in clinicopathological correlation so that appropriate advice can be given in genetic counselling. © 2013 Royal College of Pathologists of Australasia.

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    DO - 10.1097/PAT.0000000000000023

    M3 - Article

    VL - 46

    SP - 46

    EP - 52

    JO - Pathology

    JF - Pathology

    SN - 0031-3025

    IS - 1

    ER -