Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin Gene

Abstract

A broad mutational spectrum in the dystrophin (DMD) gene, from large deletions/duplications to point mutations, causes Duchenne/Becker muscular dystrophy (D/BMD). Comprehensive genotyping is particularly relevant considering the mutation-centered therapies for dystrophinopathies. We report the genetic characterization of a patient with disease onset at age 13 years, elevated creatine kinase levels and reduced dystrophin labeling, where multiplex-ligation probe amplification (MLPA) and genomic sequencing failed to detect pathogenic variants. Bioinformatic, transcriptomic (real time PCR, RT-PCR), and genomic approaches (Southern blot, long-range PCR, and single molecule real-time sequencing) were used to characterize the mutation. An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene. This corresponded to the partial exonization of a long interspersed nuclear element (LINE-1), disrupting the open reading frame. Further characterization identified a complete LINE-1 (~6 kb with typical hallmarks) deeply inserted in intron 51. Haplotyping and segregation analysis demonstrated that the mutation had a de novo origin. Besides underscoring the importance of mRNA studies in genetically unsolved cases, this is the first report of a disease-causing fully intronic LINE-1 element in DMD, adding to the diversity of mutational events that give rise to D/BMD.The authors are grateful to the patient and his family for accepting to collaborate in this work. The authors also wish to thank Dr. Stefan White and Yavuz Ariyurek from the Leiden University Medical Center (Netherlands) for facilitating the Single Molecule Real Time (PacBio) sequencing work. A research grant was attributed to J.O. by “Fundo para a Investigação e Desenvolvimento do Centro Hospitalar do Porto” (Grant ref.: 336-13(196-DEFI/285-CES)). The work was also supported by the authors’ Institutions and in part by UMIB, which is funded by “Fundação para a Ciência e Tecnologia (FCT)” under the Pest-OE/SAU/UI0215/2014.info:eu-repo/semantics/publishedVersio