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The enlargement of the genome size and the decrease in genome compactness with increase in the number and size of introns is a general pattern during the evolution of eukaryotes. Among the possible mechanisms for modifying intron size, it has been suggested that the insertion of transposable elements might have an important role in driving intron evolution. The analysis of large portions of the human genome demonstrated that a relatively recent (50 to 100 MYA) accumulation of transposable elements appears to be biased, favoring a preferential insertion of LINE1 transposons into sex chromosomes rather than into autosomes. In the present work, the effect of chromosomal location on the increase in size of introns was evaluated with a comparative analysis performed on pairs of human paralogous genes, one located on the X chromosome and the second on an autosome. A phylogenetic analysis was also performed on the X-encoded proteins and their paralogs to confirm orthology-paralogy and to approximately estimate the time of gene duplication. Statistical analysis of total intron length for each pair of paralogous genes provided no evidence for a larger size of introns in the gene copies located on the X chromosome. On the opposite, introns of autosomal genes were found to be significantly longer than introns of their X-linked paralogs. Likewise, LINE1 elements were not significantly more frequent in X-chromosome introns, whereas the frequency of SINE elements showed a marginally significant bias toward autosomal introns.

We characterised a dystrophin gene rearrangement in a previously described family with X-linked dilated cardiomyopathy and we demonstrated that it represents an 11 kb deletion occurring within intron 11. This unique deletion joined two physiologically distant intronic regions and brought adjacent two cryptic splice sites, generating a 159 bp sequence recognised as a novel alternative exon and spliced into the dystrophin transcript. Comparative analysis of the intronic region involved in the breakpoint revealed the presence of a LINE1 element (L1P_MA2), containing a 5' unconventional region (L1M1_5). This region provides the 5' cryptic splice site utilised by the novel exon, includes part of the region spliced into the dystrophin transcript and contains two short GA rich regions compatible with splicing motifs. We performed an in vitro splicing assay by using a minigene containing the patient minimal genomic rearrangement and we reproduced the inclusion of the novel alternative exon seen in the patient tissues. Antisense splicing modulation targeting the 3' cryptic splice site succeeded in restoring the canonical splicing. This represents a novel intronic mutational mechanism affecting the dystrophin gene and generating a splicing pathology. The definition of this mechanism might open perspectives in unravelling splicing regulatory motifs and their involvement in human genetic diseases.

The region of the dystrophin gene containing introns 45-50 is characterized by a high rate of recombination events that give rise to large deletions causing dystrophinopathy. The nucleotide sequence of this intronic region has recently been released in GenBank. With the aim of further understanding the mechanism favoring the occurrence of these deletions, we have characterized the distribution of introns 47 and 48 deletion endpoints in 39 dystrophinopathy patients. In 14 of these patients we were able to sequence the break junction. On these sequences we were able to identify several intronic motifs that could predispose to DNA double-strand breaks. Our results, combined with other literature data, show that unequal homologous recombination is a very poorly represented event in the dystrophin gene, whereas junction features are suggestive of a model of recombination in which DNA double-strand breaks are incorrectly repaired by a nonhomologous end-joining mechanism. The correlation among recombination rate, deletion frequency, and percentage of repetitive elements is discussed.

Over 60% of Duchenne and Becker muscular dystrophies are caused by deletions spanning tens or hundreds of kilobases in the dystrophin gene. The molecular mechanisms underlying the loss of DNA at this genomic locus are not yet understood. By studying the distribution of deletion breakpoints at the genomic level, we have previously shown that intron 49 exhibits a higher relative density of breakpoints than most dystrophin introns. To determine whether the mechanisms leading to deletions in this intron preferentially involve specific sequence elements, we sublocalized 22 deletion endpoints along its length by a polymerase-chain-reaction-based approach and, in particular, analyzed the nucleotide sequences of five deletion junctions. Deletion breakpoints were homogeneously distributed throughout the intron length, and no extensive homology was observed between the sequences adjacent to each breakpoint. However, a short sequence able to curve the DNA molecule was found at or near three breakpoint junctions.