Figure 6

Patient XL: An intronic nucleotide substitution activates a cryptic splice site leading to pseudoexon creation. The mutation is localized in intron 65 (see schematic representation of the DMD gene). A. Sequence analysis. The electropherogram depicts the insertion of 53 bp between exons 65 and 66. This insertion creates a stop codon (TGA, red box) and results in the production of a truncated protein (p.Thr3188ThrfsX7). The insertion is determined by a nucleotide substitution (c.9564-426T>G) in IVS 65, which activates a cryptic splice site. The schematic representation of IVS65 under the electropherogram depicts the position of the intronic mutation and the sequence inserted into intron 65. B. cDNA analysis depicts a band with higher molecular weight than control (Ctr) in the patient, demonstrating the insertion of 53 bp. C. Bioinformatic analysis: 5' Splice Site score distribution in patient (mutated, green) versus control (wild type, blue). The mutation is localized in a silent splice site consensus sequence included in the intron and increases the MaxENT score, that gives the probability that a sequence is a functional splice site, from a value of 6.8 to a value of 10.4. The obtained maxENT scores, suggest that the mutation activates a cripitc 5'ss leading to inclusion of the exon in the mature transcript. The substitution involves one of the most important and conserved bases of the consensus sequence, as shown in the schematic representation on the right, which illustrates the relative frequency of different bases in the 5' splice site consensus sequence (the size of the letter indicating each nucleotide reflects its importance in the consensus sequence).