Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals.

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The Complete Sequence of Dystrophin Predicts a Rod-Shaped Cytoskeletal Protein

The X-linked muscle-wasting disease Duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin. There is currently no effective treatment for the disease; however, the complex molecular pathology of this disorder is now being unravelled. Dystrophin is located at the muscle sarcolemma in a membrane-spanning protein complex that connects the cytoskeleton to the basal lamina. Mutations in many components of the dystrophin protein complex cause other forms of autosomally inherited muscular dystrophy, indicating the importance of this complex in normal muscle function. Although the precise function of dystrophin is unknown, the lack of protein causes membrane destabilization and the activation of multiple pathophysiological processes, many of which converge on alterations in intracellular calcium handling. Dystrophin is also the prototype of a family of dystrophin-related proteins, many of which are found in muscle. This family includes utrophin and α-dystrobrevin, which are involved in the maintenance of the neuromuscular junction architecture and in muscle homeostasis. New insights into the pathophysiology of dystrophic muscle, the identification of compensating proteins, and the discovery of new binding partners are paving the way for novel therapeutic strategies to treat this fatal muscle disease. This review discusses the role of the dystrophin complex and protein family in muscle and describes the physiological processes that are affected in Duchenne muscular dystrophy.

Function and genetics of dystrophin and dystrophin-related proteins in muscle

During the last quarter of the twentieth century, our knowledge about human genetic variation was limited mainly to the heterochromatin polymorphisms, large enough to be visible in the light microscope, and the single nucleotide polymorphisms (SNPs) identified by traditional PCR-based DNA sequencing. In the past five years, the rapid development and expanded use of microarray technologies, including oligonucleotide array comparative genomic hybridization and SNP genotyping arrays, as well as next-generation sequencing with “paired-end” methods, has enabled a whole-genome analysis with essentially unlimited resolution. The discovery of submicroscopic copy-number variations (CNVs) present in our genomes has changed dramatically our perspective on DNA structural variation and disease. It is now thought that CNVs encompass more total nucleotides and arise more frequently than SNPs. CNVs, to a larger extent than SNPs, have been shown to be responsible for human evolution, genetic diversity between individuals,...

Structural Variation in the Human Genome and its Role in Disease

Duchenne muscular dystrophy (DMD) and the less severe Becker muscular dystrophy (BMD) are human X-linked muscle-wasting disorders that have been localized to the band Xp21 by genetic linkage analysis1–9 and cytologically detectable abnormalities10–12. A cloned DNA segment, DXS164 (or pERT87), has been shown to detect deletions in the DNA of unrelated DMD and BMD males13–15. Here we present the nucleotide sequence of two highly conserved DNA fragments from the DXS164 locus and their homologous sequences from the mouse X chromosome. One of the human conserved segments hybridized to a large transcript in RNA isolated from human fetal skeletal muscle and was used to isolate cDNA clones which cover approximately 10% of this transcript. The cDNA clones map to Xp21 and hybridize with a minimum of eight small regions that span 130 kilobases (kb) of the DXS164 locus. These expressed sequences are candidates for portions of the gene responsible for both DMD and BMD.

Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene

We are reporting a male patient who suffered from chronic granulomatous disease associated with cytochrome b−245 deficiency and McLeod red cell phenotype, Duchenne muscular dystrophy, and retinitis pigmentosa. On cytogenetic analysis, he seemed to have a very subtle interstitial deletion of part of band Xp21. Since it was impossible to know whether this material was truly deleted or inserted elsewhere in the genome, somatic cell and molecular studies were carried out. In somatic cell hybrids, the deleted X chromosome was isolated on a Chinese hamster background. Southern blot analysis with 20 single-copy probes, that had been mapped to the X short arm, led to the discovery of one (probe 754) that is missing from this patient's X chromosome and also from his total DNA. This proves that he, indeed, has a deletion rather than a balanced insertion. The results provide cytological mapping information for the X-linked phenotypes present in this patient. Furthermore, probe 754 recognizes a restriction fragment length polymorphism of high frequency that makes it the most powerful probe currently available for linkage studies with X-linked muscular dystrophy.

Minor Xp21 chromosome deletion in a male associated with expression of duchenne muscular dystrophy, chronic granulomatous disease, retinitis pigmentosa, and McLeod syndrome

Panels of somatic cell hybrid lines carrying various structural rearrangements of the human X chromosome short arm were analyzed with 21 X-chromosome-specific cloned DNA fragments We mapped these molecular markers to five different regions of the short arm of the X chromosome The results were confirmed by gene-dosage studies of human lymphoblasts with structurally abnormal X chromosomes The ornithine transcarbamylase gene and four anonymous DNA sequences map within band Xp21, flanking the presumed locus for Duchenne muscular dystrophy

Localization of DNA sequences in region Xp21 of the human X chromosome: Search for molecular markers close to the duchenne muscular dystrophy locus

Human phosphofructokinase (PFK; EC 2.7.1.11) is under the control of three structural loci which encode muscle-type (M), live-type (L), and platelet-type (P) subunits; human diploid fibroblasts and leukocytes express all three loci. In order to assign human PFKM locus to a specific chromosome we have analyzed human x Chinese hamster somatic cell hybrids for the expression of human M subunits, using an anti-human M subunit-specific mouse monoclonal antibody. In 18 of 19 hybrids studied, the expression of the PFKM locus segregated concordantly with the presence of chromosome 1 (discordance rate 0.05) as indicated by chromosome and isozyme marker analysis. The discordance rates for all the other chromosomes were 0.32 or greater, indicating that the PFKM locus is on chromosome 1. For the regional mapping of PFKM, eight hybrids were studied that contained one of five distinct regions of chromosome 1. These results further localize the human PFKM locus to region cen leads to q32 chromosome 1.

Assignment of the human gene for muscle-type phosphofructokinase (PFKM) to chromosome 1 (region cen leads to q32) using somatic cell hybrids and monoclonal anti-M antibody.

de Martinville B

Papers

Minor Xp21 chromosome deletion in a male associated with expression of duchenne muscular dystrophy, chronic granulomatous disease, retinitis pigmentosa, and McLeod syndrome

Localization of DNA sequences in region Xp21 of the human X chromosome: Search for molecular markers close to the duchenne muscular dystrophy locus

Assignment of the human gene for muscle-type phosphofructokinase (PFKM) to chromosome 1 (region cen leads to q32) using somatic cell hybrids and monoclonal anti-M antibody.