• Open Access

The deep intronic c.903+469T>C mutation in the MTRR gene creates an SF2/ASF binding exonic splicing enhancer, which leads to pseudoexon activation and causes the cblE type of homocystinuria

Authors

  • Katerina Homolova,

    1. Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague 2, Czech Republic
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  • Petra Zavadakova,

    1. Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague 2, Czech Republic
    2. Department of Medical Genetics, University of Lausanne, Lausanne, Switzerland
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  • Thomas Koed Doktor,

    1. Research Unit for Molecular Medicine and Institute of Human Genetics, Aarhus University, Aarhus N, Denmark
    2. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark
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  • Lisbeth Dahl Schroeder,

    1. Research Unit for Molecular Medicine and Institute of Human Genetics, Aarhus University, Aarhus N, Denmark
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  • Viktor Kozich,

    1. Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague 2, Czech Republic
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  • Brage S. Andresen

    Corresponding author
    1. Research Unit for Molecular Medicine and Institute of Human Genetics, Aarhus University, Aarhus N, Denmark
    2. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark
    • Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230, Odense M, Denmark
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  • Communicated by Peter K. Rogan

Abstract

Deep intronic mutations are often ignored as possible causes of human diseases. A deep intronic mutation in the MTRR gene, c.903+469T>C, is the most frequent mutation causing the cblE type of homocystinuria. It is well known to be associated with pre-mRNA missplicing, resulting in pseudoexon inclusion; however, the pathological mechanism remains unknown. We used minigenes to demonstrate that this mutation is the direct cause of MTRR pseudoexon inclusion, and that the pseudoexon is normally not recognized due to a suboptimal 5′ splice site. Within the pseudoexon we identified an exonic splicing enhancer (ESE), which is activated by the mutation. Cotransfection and siRNA experiments showed that pseudoexon inclusion depends on the cellular amounts of SF2/ASF and in vitro RNA-binding assays showed dramatically increased SF2/ASF binding to the mutant MTRR ESE. The mutant MTRR ESE sequence is identical to an ESE of the alternatively spliced MST1R proto-oncogene, which suggests that this ESE could be frequently involved in splicing regulation. Our study conclusively demonstrates that an intronic single nucleotide change is sufficient to cause pseudoexon activation via creation of a functional ESE, which binds a specific splicing factor. We suggest that this mechanism may cause genetic disease much more frequently than previously reported. Hum Mutat 30:1–8, 2010. © 2010 Wiley-Liss, Inc.

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