Human Mutation RESEARCH ARTICLE Exon Skipping-Mediated Dystrophin Reading Frame Restoration for Small Mutations Pietro Spitali, 1y Paola Rimessi, 1y Marina Fabris, 1 Daniela Perrone, 2,3 Sofia Falzarano, 1 Matteo Bovolenta, 1 Cecilia Trabanelli, 1 Lara Mari, 2 Elena Bassi, 1 Sylvie Tuffery, 4 Francesca Gualandi, 1 Nadir M. Maraldi, 5 Patrizia Sabatelli-Giraud, 5 Alessandro Medici, 3 Luciano Merlini, 1 and Alessandra Ferlini 1Ã 1 Department of Experimental and Diagnostic Medicine, Section of Medical Genetics, University of Ferrara, Italy; 2 Department of Chemistry, University of Ferrara, Ferrara, Italy; 3 Department of Biology and Evolution, University of Ferrara, Ferrara, Italy; 4 Universite´Montpellier 1,Faculte´ de Me´decine and Inserm, U827, Montpellier, F-34000, France; 5 Institute of Anatomy, University of Bologna and IGM-CNR., Unit of Bologna, c/o IOR, Bologna, Italy Communicated by Christophe Be ´ roud Received 17 March 2009; accepted revised manuscript 30 June 2009. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/humu.21092 ABSTRACT: Exon skipping using antisense oligonucleo- tides (AONs) has successfully been used to reframe the mRNA in various Duchenne muscular dystrophy patients carrying deletions in the DMD gene. In this study we tested the feasibility of the exon skipping approach for patients with small mutations in in-frame exons. We first identified 54 disease-causing point mutations. We selected five patients with nonsense or frameshifting mutations in exons 10, 16, 26, 33, and 34. Wild-type and mutation specific 2 0 OMePS AONs were tested in cell-free splicing assays and in cultured cells derived from the selected patients. The obtained results confirm cell-free splicing assay as an alternative system to test exon skipping propensity when patients’ cells are unavailable. In myogenic cells, similar levels of exon skipping were observed for wild-type and mutation specific AONs for exons 16, 26, and 33, whereas for exon 10 and exon 34 the efficacy of the AONs was significantly different. Interestingly, in some cases skipp- ing efficiencies for mutated exons were quite dissimilar when compared with previous reports on the respective wild-type exons. This behavior may be related to the effect of the mutations on exon skipping propensity, and highlights the complexity of identifying optimal AONs for skipping exons with small mutations. Hum Mutat 30:1–8, 2009. & 2009 Wiley-Liss, Inc. KEY WORDS: exon skipping; antisense oligonucleotides; dystrophin; DMD Introduction Nonsense or frame-shifting mutations in the DMD gene (MIM] 310377) lead to Duchenne muscular dystrophy (DMD; MIM] 310200), a severe X-linked neuromuscular disorder due to the complete absence of functional dystrophin protein in males. By contrast, the milder allelic Becker muscular dystrophy (BMD; MIM] 310376) is caused by in-frame mutations that give rise to a smaller but functional protein [Hoffman et al., 1987; Monaco, 1989]. The interruption or maintenance of the dystrophin reading frame by the gene mutations explains the phenotypic differences observed in approximately 92% of the BMD/DMD cases [Aartsma-Rus et al., 2006; Koenig et al., 1989]. Although the majority of the mutations in the DMD gene consists of large rearrangements, an increasing number of both nonsense and small frame-shifting mutations has been widely reported, due to the improvement of the diagnostic systems [Bennett et al., 2001; Buzin et al., 2005; Flanigan et al., 2003; Hofstra et al., 2004; Roberts et al., 1993, 1994; Tuffery-Giraud et al., 2004; Whittock et al., 1997]. It is now estimated that point mutations account for nearly 30% of dystrophin mutations [Aartsma-Rus et al., 2006; Deburgrave et al., 2007]. Due to the approach of mutation-specific clinical trials, dystrophin mutation characterization meanwhile has become mandatory. For instance, only patients carrying nonsense mutations are eligible for participation in the phase I/IIa trial held by PTC Therapeutics, based on a stop codon read-through strategy [Welch et al., 2007]. Another mutation-specific approach is antisense- induced exon skipping aimed at the reframing of dystrophin transcripts. Among the various antisense types or modifications, the 2 0 OMePS antisense oligonucleotides (AONs) targeting exon 51 have been recently used in a pilot study in four DMD patients with very encouraging results [van Deutekom et al., 2007]. Because the majority of DMD-causing mutations are out-of- frame deletions clustered within the two major hotspot regions, the exon skipping approach has been focused on this type of rearrangements. However, it has recently been estimated that, in theory, single and double exon skipping would be applicable to 79% of deletions, 91% of small mutations, and 73% of duplications, amounting to 83% of all DMD mutations [Aartsma- Rus et al., 2009]. Indeed, only two nonsense mutations were approached in vitro by 2 0 OMePS AONs designed on the wild-type exon sequence [Aartsma-Rus et al., 2003, 2004]. OFFICIAL JOURNAL www.hgvs.org & 2009 WILEY-LISS, INC. Additional Supporting Information may be found in the online version of this article. y The first two authors contributed equally to this article. Ã Correspondence to: Alessandra Ferlini, Department of Experimental and Diagnostic Medicine, Section of Medical Genetics, University of Ferrara, 74 44100 Ferrara, Italy. E-mail: fla@unife.it