Intronic long interspersed nuclear element (LINE-1) insertion in the DMD gene as a cause of Becker Muscular Dystrophy

Long interspersed nuclear elements (LINE-1 or L1) arethe most abundant retrotransposable elements accountingfor nearly 17% of the human genome. These elementscan be randomly incorporated in the genome, thereforehaving an important role in its plasticity and in generatingstructural genetic variants. It has been demonstrated thatL1 retrotransposon activity may occasionally cause geneticdiseases. To date, only four disease-causing L1 elementshave been described in the dystrophin (DMD) gene; threeinserted in exons 44, 48 and 67, in patients with a Duchennemuscular dystrophy (DMD) phenotype, and one detectedin the 5Žuntranslated region, in two apparently unrelatedJapanese families with X-linked dilated cardiomyopathy.We report a 48 year old man with a clinical diagnosisof Becker muscular dystrophy (BMD), in 2001, withoutmolecular confirmation by multiplex PCR and Southern-Blotanalysis, and whose diagnosis was recently revisited becausehis daughter is considering pregnancy. A second molecularstudy, resorting to multiplex ligation-probe amplification(MLPA) analysis and genomic DMD gene sequencing, againfailed to detect abnormalities. A new muscle biopsy showeddystrophic features with irregular labeling for dystrophin onimmunohistochemical analysis, suggesting dystrophinopathy.With the intention of unveiling a genetic defect thatmight be refractory to the previous diagnostic techniques,muscle-derived DMD transcripts were sequenced in theirentirety. Results revealed an insertion of 103 nucleotidesbetween exons 51 and 52, which showed no homology to thegenes reference sequence. Extensive bioinformatic analysis(homology search and splice-site/branch-point analysis) andsequential direct sequencing enabled the discovery of a deepintronicinsertion of an L1 element, in intron 51. This extremelyrare mutational event resulted in the partial exonization of theL1 plus 5 nucleotides of intron 51. In addition to the aberrant out-of-frame transcript, a residually expressed wild-typetranscript was also detected, thereby explaining the milderphenotype in this patient.To our knowledge this is the first report ever ofdystrophinopathy caused by an intronically placed L1element. Besides representing an exceptional contributiontowards widening the DMD gene mutation spectrum, thisstudy highlights the importance of conducting mRNA studiesin as yet uncharacterized BMD/DMD patients. This holds trueeven considering some of the most recent state-of-the-artscreening approaches, based on next-generation sequencingtechnology, where this type of mutation may ultimately fail tobe detected