Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

Patient recruitment, breakpoint definition, RNA profiling, antisense modulation in patients with dystrophin mutations

We performed a prenatal diagnosis in a family with X-linked dilated cardiomyopathy (XLDC) in which the causative mutation was a pure intronic deletion which causes the splicing of a novel, aberrant, out-of-frame exon into the dystrophin transcript. Our genetic test was performed by defining both the DNA (villous) and the RNA (amniocytes) configuration. The prenatal diagnosis resulted in a female foetus, proven to be a carrier of the genomic deletion. RNA analysis on cultured amniocytes revealed the presence of a well detectable dystrophin transcript as well as the coexistence of both the wild type and the abnormal splicing profile.

This finding suggests that dystrophin splicing pattern in amniocytes and skeletal muscle is similar and that therefore this approach could be used in other prenatal dystrophin mutation detection, where abnormal RNA splicing is thought to play a role or for specific cases in which no mutations have been identified in the coding regions.

We have precisely characterized genomic breakpoints within introns 2, 6 and 7 and identified the splicing profiles in a cohort of DMD/BMD patients with deletion of exons 3-7, 3-6 and duplication of exons 2-4. The findings of our study support the possibility that the re-initiation of translation mechanism play a role in exons 3-7 deleted patients phenotypic variability. Furthermore, we observed that the out-of-frame exon 2a is constantly spliced into a proportion of the dystrophin transcripts in all patients analysed.

We identified and functionally characterised a purine-rich sequence located within dystrophin intron 11 and involved in splicing regulation. A functional role of this motif was suggested by an intronic dystrophin mutation causing X-linked dilated cardiomyopathy and determining the tissue-specific incorporation of an aberrant exon into the dystrophin transcript. This motif, as well as the 5’ cryptic splice site used by the aberrant exon in the XLDC family, is contained within an atypical LINE1 5’ region. The splicing sequence is contained within the aberrant XLDC exon and behaves in vitro as a splicing enhancer because its deletion strongly interferes with the dystrophin exon inclusion.

Furthermore, by using RNA electro-mobility shift assay (REMSA) and sequence-specific UV cross-linking, we demonstrated that the splicing motif displays a nuclear protein binding affinity, further supporting a functional role in splicing modulation.

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 crytpic 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 a GA rich region compatible with a splicing motif.

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’crytpic 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.

We have studied two Duchenne patients carrying the in-frame isolated deletion of dystrophin exon 5 and we have defined the genomic deletion breakpoints. Transcription analysis in skeletal muscle from one patient revealed a complex RNA configuration, combining an unfavorable exon skipping event (involving exon 6 and leading to an out-of-frame transcript) with the production of scrambled, circular RNA molecules. RNA circularization specifically involved the in-frame transcripts (retaining exon 6) with the consequence of depletion of the functional messenger.

In addition, we also documented abundant in-frame exon 9 skipping. This peculiar splicing behaviour leading to a defect in the in-frame messenger RNA and in critical protein domains, might represent the pathogenic background underlying the severe clinical impact of the rare exon 5 deletion. The circular molecule formation focuses attention on the role that RNA scrambling might have in contributing to the clinical severity in dystrophin deletions.


Alessandra FERLINI, (Dirigente Medico di 1° livello (Senior Consultant Hospital))
Tel.: +39-053-2424436
Fax: +39-053-2236157
Correo electrónico
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