Acute subcutaneous emphysema: A rare clinical presentation of large bowel perforation.

These atypical presentations of colonic perforation illustrate the importance of considering acute intra-abdominal pathology when subcutaneous emphysema is identified.

Laparoscopic watson fundoplication is effective and durable in children with gastrooesophageal reflux.

Gastroesophageal reflux (GOR) affects 2-8% of children over 3 years of age and is associated with significant morbidity. The disorder is particularly critical in neurologically impaired children, who have a high risk of aspiration. Traditionally, the surgical antireflux procedure of choice has been Nissen's operation. However, this technique has a significant incidence of mechanical complications and has a reoperation rate of approximately 7%, leading to the development of alternative approaches. Watson's technique of partial anterior fundoplication has been shown to achieve long-lasting reflux control in adults with few mechanical complications, but there is limited data in the paediatric population. We present here short- and long-term outcomes of laparoscopic Watson fundoplication in a series of 76 children and infants, 34% of whom had a degree of neurological impairment including severe cerebral palsy and hypoxic brain injury. The overall complication rate was 27.6%, of which only 1 was classified as major. To date, we have not recorded any incidences of perforation and no revisions. In our experience, Watson's laparoscopic partial fundoplication can be performed with minimal complications and with durable results, not least in neurologically compromised children, making it a viable alternative to the Nissen procedure in paediatric surgery.

Adenovirus-based targeting in myoblasts is hampered by nonhomologous vector integration.

Chromosomal correction of dystrophin gene mutations is a most desirable therapeutic solution for Duchenne muscular dystrophy, as it allows production of the full-length dystrophin under the control of locus-specific promoters. Here we explored gene targeting in conditionally immortal mouse dystrophin-deficient myoblasts. We constructed an adenoviral vector for the correction of the mdx mutation, containing 6.0 kb of sequence homologous to the target locus (partial intron 21 through to exon 24 with the normal sequence of exon 23) and a neomycin expression cassette inserted in intron 23. Adenovirus-based gene targeting was previously reported to be beneficial in mouse embryonic stem cells, resulting in one targeted integration per three integration events. However, we found no targeted integration events among 144 stably transduced G418-resistant myoblast clones, reflecting efficient random integration of the adenoviral vector in myogenic cells. We found that mouse myoblasts are capable of integrating recombinant adenoviral DNA with an efficiency approaching 1%. Interestingly, dermal fibroblasts integrate adenoviral DNA up to 100 times less efficiently than myoblasts from the same mice. We also show that the efficiency of recombinant adenoviral DNA integration is influenced by preinfection cell density, possibly indicating the importance of cellular DNA replication for adenoviral integration.

Genomic definition of a pure intronic dystrophin deletion responsible for an XLDC splicing mutation: in vitro mimicking and antisense modulation of the splicing abnormality.

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.

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts.

The multiplicity of proteins compared with genes in mammals owes much to alternative splicing. Splicing signals are so subtle and complex that small perturbations may allow the production of new mRNA variants. However, the flexibility of splicing can also be a liability, and several genetic diseases result from single-base changes that cause exons to be skipped during splicing. Conventional oligonucleotide strategies can block reactions but cannot restore splicing. We describe here a method by which the use of a defective exon was restored. Spinal muscular atrophy (SMA) results from mutations of the Survival Motor Neuron (SMN) gene. Mutations of SMN1 cause SMA, whereas SMN2 acts as a modifying gene. The two genes undergo alternative splicing with SMN1, producing an abundance of full-length mRNA transcripts, whereas SMN2 predominantly produces exon 7-deleted transcripts. This discrepancy is because of a single nucleotide difference in SMN2 exon 7, which disrupts an exonic splicing enhancer containing an SF2ASF binding site. We have designed oligoribonucleotides that are complementary to exon 7 and contain exonic splicing enhancer motifs to provide trans-acting enhancers. These tailed oligoribonucleotides increased SMN2 exon 7 splicing in vitro and rescued the incorporation of SMN2 exon 7 in SMA patient fibroblasts. This treatment also resulted in the partial restoration of gems, intranuclear structures containing SMN protein that are severely reduced in patients with SMA. The use of tailed antisense oligonucleotides to recruit positively acting factors to stimulate a splicing reaction may have therapeutic applications for genetic disorders, such as SMA, in which splicing patterns are altered.