Figure 4

DNA and RNA analysis in patients 10, 8 and 9. a) RT-PCR analysis: the amplification of exons 52–57, 63–68, 35–40 respectively, resulted in fragments larger than controls. b) Right: direct sequencing of the exons 52–57 amplification product in patient 10 showed an insertion of 50 bp (upper box) between exons 55 and 56, derived from intron 55; Left: sequence of the intron 55 specific region showed an A to G transition that lead to the creation of an acceptor splice site; c) Right: direct sequencing of the exons 63–68 amplification product in patient 8 showed an insertion of 147 bp (upper box) derived from intron 65 between exons 65 an 66; Left: sequence of the intron 65 specific region showed an A to G transition in intron 65 that result in the creation of a donor splice site; d) Right: direct sequencing of the exons 35–40 amplification product in patient 9 showed an insertion of 77 bp (upper box) derived from intron 37 between exons 37 and 38; Left: the genomic sequence surrounding the intron 37 region inserted into the mature transcript revealed the presence of a canonical acceptor and donor splice sites flanking the sequence itself. Moreover we identified a deletion of 18 nucleotides (reported in the box) which is located 20 nucleotides upstream in respect to the sequence inserted into the transcript.