Duchenne and Becker muscular dystrophies are caused by defects of the dystrophin gene. Expression of this large X-linked gene is under elaborate transcriptional and splicing control. At least five independent promoters specify the transcription of their respective alternative first exons in a cell-specific and developmentally controlled manner. Three promoters express full-length dystrophin, while two promoters near the C terminus express the last domains in a mutually exclusive manner. Six exons of the C terminus are alternatively spliced, giving rise to several alternative forms. Genetic, biochemical and anatomical studies of dystrophin suggest that a number of distinct functions are subserved by its great structural diversity. Extensive studies of dystrophin may lead to an understanding of the cause and perhaps a rational treatment for muscular dystrophy.

The structural and functional diversity of dystrophin

We have constructed a 3.1 megabase (Mb) physical map of chromosome 17p11.2-p12, which contains a submicroscopic duplication in patients with Charcot-Marie-Tooth disease type 1A (CMT1A). We find that the CMT1A duplication is a tandem repeat of 1.5 Mb of DNA. A YAC contig encompassing the CMT1A duplication and spanning the endpoints was also developed. Several low copy repeats in 17p11.2-p12 were identified including the large (> 17 kb) CMT1A-REP unit which may be part of a mosaic repeat. CMT1A-REP flanks the 1.5 Mb CMT1A monomer unit on normal chromosome 17 and is present in an additional copy on the CMT1A duplicated chromosome. We propose that the de novo CMT1A duplication arises from unequal crossing over due to misalignment at these CMT1A-REP repeat sequences during meiosis.

Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit.

UMD-DMD France is a knowledgebase developed through a multicenter academic effort to provide an up-to-date resource of curated information covering all identified mutations in patients with a dystrophinopathy The current release includes 2,411 entries consisting in 2,084 independent mutational events identified in 2,046 male patients and 38 expressing females, which corresponds to an estimated number of 39 people per million with a genetic diagnosis of dystrophinopathy in France Mutations consist in 1,404 large deletions, 215 large duplications, and 465 small rearrangements, of which 398% are nonsense mutations The reading frame rule holds true for 96% of the DMD patients and 93% of the BMD patients Quality control relies on the curation by four experts for the DMD gene and related diseases Data on dystrophin and RNA analysis, phenotypic groups, and transmission are also available About 24% of the mutations are de novo events This national centralized resource will contribute to a greater understanding of prevalence of dystrophinopathies in France, and in particular, of the true frequency of BMD, which was found to be almost half (43%) that of DMD UMD-DMD is a searchable anonymous database that includes numerous newly developed tools, which can benefit to all the scientific community interested in dystrophinopathies Dedicated functions for genotype-based therapies allowed the prediction of a new multiexon skipping (del 45-53) potentially applicable to 53% of the deleted DMD patients Finally, such a national database will prove to be useful to implement the international global DMD patients' registries under development

Genotype-phenotype analysis in 2,405 patients with a dystrophinopathy using the UMD-DMD database: a model of nationwide knowledgebase.

Aminoglycosides have previously been shown to suppress nonsense mutations, allowing translation of full-length proteins in vitro and in animal models. In the mdx mouse, where muscular dystrophy is due to a nonsense mutation in the dystrophin gene, gentamicin suppressed truncation of the protein and ameliorated the phenotype. A subset of patients with Duchenne and Becker muscular dystrophy similarly possess a nonsense mutation, causing premature termination of dystrophin translation. Four such patients, with various stop codon sequences, were treated once daily with intravenous gentamicin at 7.5 mg/kg/day for 2 weeks. No ototoxicity or nephrotoxicity was detected. Full-length dystrophin was not detected in pre- and post-treatment muscle biopsies.

Gentamicin treatment of Duchenne and Becker muscular dystrophy due to nonsense mutations

The spinal muscular atrophy (SMA) region on chromosome 5q13 contains an inverted duplication of about 500 kb, and deleterious mutations in the survival motor neuron 1 (SMN1) gene cause SMA, a common lethal childhood neuropathy. We have used a number of approaches to probe the evolutionary history of these genes and show that SMN gene duplication and the appearance of SMN2 occurred at very distinct evolutionary times. Molecular fossil and molecular clock data suggest that this duplication may have occurred as recently as 3 million years ago in that the position and identity repetitive elements are identical for both human SMN genes and overall sequence divergence ranged from 0.15% to 0.34%. However, these approaches ignore the possibility of sequence homogenization by means of gene conversion. Consequently, we have used quantitative polymerase chain rection and analysis of allelic variants to provide physical evidence for or against SMN gene duplication in the chimpanzee, mankind's closest relative. These studies have revealed that chimpanzees have 2-7 copies of the SMN gene per diploid genome; however, the two nucleotides diagnostic for exons 7-8 and the SMNdelta7 mRNA product of the SMN2 gene are absent in non-human primates. In contrast, the SMN2 gene has been detected in all extant human populations studied to date, including representatives from Europe, the Central African Republic, and the Congo. These data provide conclusive evidence that SMN gene duplication occurred more than 5 million years ago, before the separation of human and chimpanzee lineages, but that SMN2 appears for the first time in Homo sapiens.

SMN gene duplication and the emergence of the SMN2 gene occurred in distinct hominids: SMN2 is unique to Homo sapiens

Partial gene deletion is the major cause of mutation leading to Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) Partial gene duplication has also been recognized in a few cases We have conducted a survey for duplication in 72 unrelated nondeletion patients, analyzed by Southern blot hybridization with clones representing the entire DMD cDNA With careful quantitative analysis of hybridization band intensity, 10 cases were found to carry a duplication of part of the gene, a frequency of 14% for nondeletion cases (10/72), or 6% for all cases (10/181) The extent of these duplications has been characterized according to the published exon-containing HindIII fragment map, and in six of the 10 duplications a novel restriction fragment that spanned the duplication junction was detected The resulting translational reading frame of mRNA has been predicted for nine duplications A shift of the reading frame was predicted in four of the six DMD cases and in one of the two intermediate cases, while the reading frame remained uninterrupted in both BMD cases RFLP and quantitative Southern blot analyses revealed a grandpaternal origin of duplication in four families and grandmaternal origin in one family In all five families, the duplication was found to originate from a single X chromosome Unequal sister-chromatid exchange is proposed to be the mechanism for the formation of these duplications

Xiuyuang Hu

Papers

Duplicational mutation at the Duchenne muscular dystrophy locus: its frequency, distribution, origin, and phenotypegenotype correlation.