Duchenne muscular dystrophy.

Progress in understanding the role of dystrophin raises promising hopes for a treatment for Duchenne muscular dystrophy. In addition, great improvements have been made in the ability to diagnose this disease using simple molecular methods.     

Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification.

The application of recombinant DNA technology to prenatal diagnosis of many recessively inherited X-linked diseases is complicated by a high frequency of heterogeneous, new mutations (1). Partial gene deletions account for more than 50% of Duchenne muscular dystrophy (DMD) lesions, and approximately one-third of all cases result from a new mutation (2-5). We report the isolation and DNA sequence of several deletion prone exons from the human DMD gene. We also describe a rapid method capable of detecting the majority of deletions in the DMD gene. This procedure utilizes simultaneous genomic DNA amplification of multiple widely separated sequences and should permit deletion scanning at any hemizygous locus. We demonstrate the application of this multiplex reaction for prenatal and postnatal diagnosis of DMD.     

Duchenne muscular dystrophy: deficiency of dystrophin at the muscle cell surface

Dystrophin is the altered gene product in Duchenne muscular dystrophy (DMD). We used polyclonal antibodies against dystrophin to immunohistochemically localize the protein in human muscle. In normal individuals and in patients with myopathies other than DMD, dystrophin was localized to the sarcolemma of the fibers. The protein was absent or markedly deficient in DMD. The sarcolemmal localization of dystrophin is consistent with other evidence that there are structural and functional abnormalities of muscle surface membranes in DMD.     

Accurate assessment of intragenic recombination frequency within the Duchenne muscular dystrophy gene

Polymorphic loci that lie at the two extremities of the Duchenne/Becker muscular dystrophy (DMD/BMD) gene have been used to estimate intragenic recombination rates. Multipoint linkage analysis of the CEPH panel of families suggests a total intragenic recombination frequency of nearly 0.12 (confidence intervals 0.041-0.226) over the genomic length of approximately 2 Mb.     

Evidence for mutation by unequal sister chromatid exchange in the Duchenne muscular dystrophy gene.

We have studied three families each containing a male with Duchenne or Becker muscular dystrophy. Southern blot analysis using both genomic and cDNA probes revealed that an exon-containing segment of DNA within the gene is duplicated in the probands, their mothers, and, in two cases, their sisters. The grandpaternal origin of the duplication has been demonstrated in these families by RFLP and duplication analysis. The results suggest that unequal sister-chromatid exchange, which most likely occurred in the germ cell lineage of the proband's grandfather, is responsible for generating these duplications and that this type of intrachromosomal rearrangement, although rarely reported in humans, is not uncommon in the muscular dystrophy gene.     

Creatine kinase, cell membrane and Duchenne muscular dystrophy

In 1958 Professor Setsuro Ebashi found that serum creatine kinase activity is increased in patients suffering from various muscular dystrophies, especially Duchenne muscular dystrophy (DMD). He and others proposed that creatine kinase passes through the cell membrane as it is released from DMD muscle fibers.Since then, it has been found that dystrophin and dystrophin-associated proteins are connected to several other components, including the basal lamina and subsarcolemmal cytoskeletal networks on the cell membrane, while dystrophin anchors these dystrophin-associated proteins to the actin filaments inside the muscle cell. In DMD muscle, dystrophin has been found to be absent and dystroglycans and sarcoglycans decreased. However, how creatine kinase molecules can pass through the DMD muscle cell membrane still remains unanswered.On the basis of recent findings on the structure of the protein layers which sandwich the lipid bilayer of muscle cell membranes, this essay stresses the importance of these lipid bilayers in protecting creatine kinase release from protoplasma in normal muscle. It further indicates the possibility that the absence of dystrophin in DMD muscle during muscle contraction may result in temporal damage to the lipid bilayer.     

Human X chromosome markers and Duchenne muscular dystrophy.

Two DNA markers, a random DNA fragment 754 and the cDNA sequence encoding the gene for ornithine transcarbamylase (OTC) have been studied in kindreds segregating for Duchenne muscular dystrophy. 754 and OTC are located close physically to the mutation in the region Xp21 below the breakpoints in two Duchenne females. The genetic distance was found to be approximately 10cM between 754 and DMD (two crossovers in 26 meioses) and to be approximately 10cM between OTC and DMD (two crossovers in 26 meioses). Physical data suggest the order DMD-754-OTC. The frequency of recombination compared to physical distance between these markers and DMD suggests that there may be a hot spot of recombination. The relevance of these observations for the isolation of the DMD mutation and clinical use of these probes is discussed.     

Germinal mosaicism from grand-paternal origin in a family with Duchenne muscular dystrophy

Summary We have identified a Duchenne muscular dystrophy (DMD) pedigree with an unexpected pattern of inheritance. Using marker restriction fragment length polymorphisms detected by probes that lie within and outside the DMD gene, we could demonstrate that the maternal grandfather has transmitted two distinct types of X chromosomes to his offspring. This original observation may be explained by postulating that the DMD mutation must have occurred during mitosis in early germline proliferation, leading to a germline mosaicism within this male ancestor.     

Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy

CONTEXT:

Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders.

AIM:

To study the utility of MLPA in diagnosis and carrier detection for DMD.

MATERIALS AND METHODS:

Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared.

RESULTS AND CONCLUSIONS:

We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.     

Duchenne muscular dystrophy and dystrophin: Sequence homology observations

Duchenne muscular dystrophy (DMD) is a genetically transmitted disease characterized by progressive muscle weakness and usually leads to death. DMD results from the absence, deficiency or dysfunction of the protein dystrophin. Analysis of protein data bases, including homology alignments and domain recognition patterns, have located highly significant correlations between dystrophin and other calcium regulating proteins. In particular, a major portion of the dystrophin sequence has been found to contain repeating units of approximately 100 amino acid residues. These repeating units were found to exhibit significant homology to troponin I. Troponin I has been found to bind to the calcium binding proteins calmodulin and troponin C. The regions of highest homology were characterized by patterns of high localization of charged amino acids and thus could represent a possible calmodulin or troponin C surface accessible binding site. Since subcellular localization studies have indicated that dystrophin is associated with the triadic junction, these findings imply that dystrophin could be involved in controlling intracellular calcium homeostasis.Special issue dedicated to Dr. Lawrence Austin.     

Estimation of the male and female mutation rates in Duchenne muscular dystrophy (DMD)

Summary We present the results of an international collaborative study aimed at estimating the ratio of male to female mutation rates in Duchenne muscular dystrophy based on the method of C. Müller and T. Grimm. With a sample size of 295, this ratio is found to be very close to 1, thus giving evidence for equal mutation rates in males and females in Duchenne muscular dystrophy.