UNLABELLED: Mutations affecting splicing underlie the development of many human genetic diseases, but rather rarely through mechanisms of pseudoexon activation. Here, we describe a novel c.1092T>A mutation in the iduronate-2-sulfatase (IDS) gene detected in a patient with significantly decreased IDS activity and a clinical diagnosis of mild mucopolysaccharidosis II form. The mutation created an exonic de novo acceptor splice site and resulted in a complex splicing pattern with multiple pseudoexon activation in the patient's fibroblasts. Using an extensive series of minigene splicing experiments, we showed that the competition itself between the de novo and authentic splice site led to the bypass of the authentic one. This event then resulted in activation of several cryptic acceptor and donor sites in the upstream intron. As this was an unexpected and previously unreported mechanism of aberrant pseudoexon inclusion, we systematically analysed and disproved that the patient's mutation induced any relevant change in surrounding splicing regulatory elements. Interestingly, all pseudoexons included in the mature transcripts overlapped with the IDS alternative terminal exon 7b suggesting that this sequence represents a key element in the IDS pre-mRNA architecture. These findings extend the spectrum of mechanisms enabling pseudoexon activation and underscore the complexity of mutation-induced splicing aberrations. KEY MESSAGE: Novel exonic IDS gene mutation leads to a complex splicing pattern. Mutation activates multiple pseudoexons through a previously unreported mechanism. Multiple cryptic splice site (ss) activation results from a bypass of authentic ss. Authentic ss bypass is due to a competition between de novo and authentic ss.

Center for Medical Genetics UZ Brussel and Vrije Universiteit Brussel 1090 Brussels Belgium

Central European Institute of Technology Masaryk University Brno Czech Republic

Institute of Clinical Immunology and Allergology St Anne's University Hospital and Masaryk University Brno Czech Republic

Institute of Inherited Metabolic Disorders 1st Faculty of Medicine Charles University Prague Czech Republic

International Centre for Genetic Engineering and Biotechnology Trieste Italy

Molecular Genetics Laboratory Centre for Cardiovascular Surgery and Transplantation Pekařská 53 656 91 Brno Czech Republic

Nutrition and Metabolism Unit University Children's Hospital Queen Fabiola Free University of Brussels 1020 Brussels Belgium

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