Use of genetically modified muscle and fat grafts to repair defects in bone and cartilage.

We report a novel technology for the rapid healing of large osseous and chondral defects, based upon the genetic modification of autologous skeletal muscle and fat grafts. These tissues were selected because they not only possess mesenchymal progenitor cells and scaffolding properties, but also can be biopsied, genetically modified and returned to the patient in a single operative session. First generation adenovirus vector carrying cDNA encoding human bone morphogenetic protein-2 (Ad.BMP-2) was used for gene transfer to biopsies of muscle and fat. To assess bone healing, the genetically modified ("gene activated") tissues were implanted into 5mm-long critical size, mid-diaphyseal, stabilized defects in the femora of Fischer rats. Unlike control defects, those receiving gene-activated muscle underwent rapid healing, with evidence of radiologic bridging as early as 10 days after implantation and restoration of full mechanical strength by 8 weeks. Histologic analysis suggests that the grafts rapidly differentiated into cartilage, followed by efficient endochondral ossification. Fluorescence in situ hybridization detection of Y-chromosomes following the transfer of male donor muscle into female rats demonstrated that at least some of the osteoblasts of the healed bone were derived from donor muscle. Gene activated fat also healed critical sized defects, but less quickly than muscle and with more variability. Anti-adenovirus antibodies were not detected. Pilot studies in a rabbit osteochondral defect model demonstrated the promise of this technology for healing cartilage defects. Further development of these methods should provide ways to heal bone and cartilage more expeditiously, and at lower cost, than is presently possible.

Retinoblastoma gene promoter directs transgene expression exclusively to the nervous system.

In human, germ line mutations in the tumor suppressor retinoblastoma (Rb) predispose individuals to retinoblastoma, whereas somatic inactivation of Rb contributes to the progression of a large spectrum of cancers. In mice, Rb is highly expressed in restricted cell lineages including the neurogenic, myogenic, and hematopoietic systems, and disruption of the gene leads to specific embryonic defects in these tissues. The symmetry between Rb expression and the defects in mutant mice suggest that transcriptional control of Rb during embryogenesis is pivotal for normal development. We have initiated studies to dissect the mechanisms of transcriptional regulation of Rb during development by promoter lacZ transgenic analysis. DNA sequences up to 6 kilobase pairs upstream of the mouse Rb promoter, isolated from two different genomic libraries, directed transgene expression exclusively to the developing nervous system, excluding skeletal muscles and liver. Expression of the transgene in the central and peripheral nervous systems, including the retina, recapitulated the expression of endogenous Rb during embryogenesis. A promoter region spanning approximately 250 base pairs upstream of the transcriptional starting site was sufficient to confer expression in the central and peripheral nervous systems. To determine whether this expression pattern was conserved, we isolated the human Rb 5' genomic region and generated transgenic mice expressing lacZ under control of a 1.6-kilobase pair human Rb promoter. The human Rb promoter lacZ mice also expressed the transgene primarily in the nervous system in several independent lines. Thus, transgene expression directed by both the human and mouse Rb promoters is restricted to a subset of tissues in which Rb is normally expressed during embryogenesis. Our findings demonstrate that regulatory elements directing Rb expression to the nervous system are delineated within a well defined core promoter and are regionally separated from elements, yet to be identified, that are required for expression of Rb in the developing hematopoietic and skeletal muscle systems.

Stillbirths in Qatar: a review of 83 cases.

To evaluate the aetiology, diagnostic procedures and current management of stillbirths in Qatar, 83 stillbirths with a birth weight of more than 500 g were studied. The validity of the cause of death was classified as certain, probable and unexplained. Frequency and descriptive statistics were used. The stillbirth rate was 8.15 per 1000. The cause of death was certain in 29%, probable in 62% and remained entirely unexplained in 9% of the cases. The major factors that might be the causes of fetal death were intrauterine growth retardation (23%), abruptio placentae (16.3%), congenital anomalies (13.3%), gestational diabetes (9.6%) and hydrops fetalis (7.2%). The cause of death was found unavoidable in 24 cases (29%). The autopsy rate was terribly low (1/80) and far away from the recommended rate of 75%. The introduction of a stillbirth programme, that includes post-mortem autopsy, in any maternity hospital, is considered crucial to reach a specific diagnosis for almost all stillbirths and to prevent fetal death in future pregnancies. However, if the patient or her family refused autopsy, a combination of patience and learned communication can pave the way to their understanding and acceptance of the procedure. Postmortem magnetic resonance imaging may be used as alternative to autopsy if it is refused.

Double missense mutation in exon 41 of the human dystrophin gene detected by double strand conformation analysis.

Development of late-onset Becker muscular dystrophy is reported in a patient whose two healthy brothers showed high serum creatine kinase level. No cases of neuromuscular disorders had been previously reported in this family. The analysis of the dystrophin gene showed that the three brothers had A-->C transversion at nucleotide 6092 in exon 41, a missense mutation which converts lysine into glutamine. The symptomatic patient showed an additional mutation in the same exon, a T-->C transition at nucleotide 6119, converting a phenylalanine to leucine. The possible pathogenic role of this mutation is discussed.

Extra-muscle involvement in dystrophinopathies: an electroretinography and evoked potential study.

Dystrophin is present in various tissues other than skeletal and cardiac muscles, including the central nervous system (CNS) and the outer plexiform layer of the retina. Therefore lack of dystrophin might be related to mental retardation or to changes in electrophysiological tests exploring retina and CNS. We performed electroretinography, VEPs, BAEPs, SEPs and MEPs in 18 patients with Duchenne muscular dystrophy (DMD), 18 with Becker muscular dystrophy (BMD) and 12 obligate carriers. We observed a marked reduction of the b-wave amplitude in the scotopic ERG, mainly in DMD patients. Oscillatory potentials were altered in all groups, even in carriers, suggesting that dystrophin may be also involved in retinal circulation. VEPs changes confirmed the role of dystrophin in visual function. The other evoked potentials were altered only in a small percentage of subjects but changes of different tests did not overlap in individual subjects. Neurophysiological abnormalities did not correlate with type, site and size of alteration in the dystrophin gene.

A study on duplications of the dystrophin gene: evidence of a geographical difference in the distribution of breakpoints by intron.

Starting from a group of 265 Italian patients affected with Duchenne or Becker muscular dystrophy a screening for duplications in the dystrophin gene was performed on 112 cases in which no deletions had previously been detected. The 21 intragenic duplications detected account for 7.9% of the total. Among these, one duplication including exons from 3 to 43 is the largest reported so far. Data from this study were combined with those from the literature and breakpoint distribution by intron was analysed. In general breakpoints occur mostly in the proximal third of the gene, in particular in intron 7. However, both the frequency of duplications and the distribution of breakpoints by intron are different in the Japanese sample compared with the other groups of patients. The role of geographical differentiation of intron sequences by genetic drift and of transposon-like sequences in explaining these differences is discussed.

A possible missense mutation detected in the dystrophin gene by Double-Strand Conformation Analysis (DSCA).

A new and simple method for detecting point mutations is presented. The method, based on Double-Strand Conformation Analysis (DSCA) of PCR amplification products in polyacrylamide gel electrophoresis, was applied to 78 unrelated subjects affected with Duchenne or Becker muscular dystrophy and to 9 subjects suspected to be affected with an atypical dystrophinopathy. An A-->G substitution in the nucleotide 2525, which changes the codon for lysine to a codon for glutamic acid was detected in an 8-year-old boy, with normal neurological examination, but showing increased CK level and an abnormal EMG. The muscle biopsy was normal, without features of necrosis or regeneration. Immunoreactions with anti-dystrophin antibodies showed a normal distribution and intensity of the staining. A review of the dystrophin mutations detected so far is included.

Prevalent cardiac involvement in dystrophin Becker type mutation.

Myocardial involvement is frequently present in Xp21-linked muscular dystrophy, due to a lack of dystrophin in cardiac fibres. We describe a 41-yr-old man affected by dilated cardiomyopathy with sporadic episodes of myoglobinuria induced by effort and increased levels of serum creatine kinase. Very mild signs of skeletal myopathy were clinically evident. His mother was affected by an indefinite cardiopathy and suddenly died when she was 36 yr old. Muscle biopsy of the patient showed a dystrophic process. Dystrophin analysis together with a genetic DMD locus study led us to diagnose Becker type muscular dystrophy, with truncated dystrophin and a gene deletion extending from exon 45 to 48. Prevalent cardiac involvement in a Becker type mutation of the dystrophin gene further confirms clinical variability of dystrophinopathies.

Dystrophin-positive myotubes in innervated muscle cultures from Duchenne and Becker muscular dystrophy patients.

Nerve-muscle co-cultures from five Duchenne muscular dystrophy (DMD) patients and one Becker (BMD) patient, were studied by immunocytochemistry with antibodies against different portions of dystrophin. Four DMD patients had a deletion in the dystrophin gene. Some dystrophin-positive myotubes were detected in a few samples of all DMD cases. PCR amplification of exon 8 of the dystrophin gene ruled out a contamination from rat spinal cord during innervation. Our results in three DMD cases, may be explained by a clonal selection of dystrophin-positive fibers observed in muscle biopsies, while in the other two cases, a "frame-restoring" mutation might account for the presence of dystrophin-positive myotubes. The possible expression of "dystrophin-related protein" or dystrophin immature isoform was considered. In the BMD case an abnormal truncated dystrophin was found in innervated muscle cultures, as well as in muscle biopsy.

Reappraisal of the incidence rate of Duchenne and Becker muscular dystrophies on the basis of molecular diagnosis.

The incidence rates of Duchenne and Becker muscular dystrophies (X-linked recessive) in a given sample of the Italian population were recalculated using the results of DNA and dystrophin analysis. While the incidence rate of Duchenne muscular dystrophy remained unchanged, the new figure for the incidence of Becker muscular dystrophy (7.2 per 100,000 male live births) was much higher than previously reported, since molecular diagnosis revealed additional cryptic cases, but this incidence is still an underestimate.