Fig. 2.

Functional analysis of DMD mutation. PCR analysis from control and DMD mRNA isolated form peripheral leukocytes was performed in order to understand if the mutation in the acceptor splice site of intron 26 (AG → AC) was responsible for uncorrect splicing and abnormal dystrophin transcript. Both samples control and DMD samples apparently produced a single band of the expected size (exons 25–29) (a). Direct sequence analyses confirmed that the band obtained by PCR corresponded to the full-length isoform of dystrophin for both control and DMD samples (b-c). Dystrophin mRNA of the DMD patient was found to miss the first two nucleotides (AG) of exon 27. The mechanism proposed is that in the presence of the mutated acceptor splice site of intron 26 (AC), the first two nucleotides of intron 27 are recognized as a new acceptor splice site due to sequence homology and are not included in the mRNA. The elimination of nucleotides AG from exon 27 generates a STOP codon in exon 27 that do not permit the production of a functional dystrophin (c)