Prenatal diagnosis of Duchenne muscular dystrophy by fetal muscle biopsy

Summary Prenatal diagnosis and carrier detection for Duchenne muscular dystrophy (DMD) usually can be performed using DNA analysis. When recombination occurs within the DMD gene, or DNA analysis is uninformative, or in pedigrees where it is unclear whether or not the consultand is a carrier, direct examination of muscle by dystrophin analysis may provide the only means of prenatal diagnosis. We present three cases representing each of these molecular genetic diagnostic dilemmas. In each instance, we used sonographically guided fetal muscle biopsy for dystrophin protein analysis to resolve the dilemma. In the first and third cases, the presence of normal dystrophin was shown by immunofluorescence and this was followed by delivery of an unaffected male fetus. In the second case, dystrophin was not found in fetal muscle tissue implying that this fetus was affected. The absence of dystrophin and affected status was confirmed in skeletal and cardiac muscle obtained after pregnancy termination.     

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.     

The molecular basis of activity-induced muscle injury in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common of the human muscular dystrophies, affecting approximately 1 in 3500 boys. Most DMD patients die in their late teens or early twenties due to involvement of the diaphragm and other respiratory muscles by the disease. The primary abnormality in DMD is an absence of dystrophin, a 427 kd protein normally found at the cytoplasmic face of the muscle cell surface membrane. Based upon the predicted structure and location of the protein, it has been proposed that dystrophin plays an important role in providing mechanical reinforcement to the sarcolemmal membrane of muscle fibers. Therefore, dystrophin could help to protect muscle fibers from potentially damaging tissue stresses developed during muscle contraction. In the present paper, the nature of mechanical stresses placed upon myofibers during various forms of muscle contraction are reviewed, along with current lines of evidence supporting a critical role for dystrophin as a subsarcolemmal membrane-stabilizing protein in this setting. In addition, the implications of these findings for exercise programs and other potential forms of therapy in DMD are discussed.     

Dietary NaCl supplementation prevents muscle necrosis in a mouse model of Duchenne muscular dystrophy

The mdx mouse is an animal model for Duchenne muscular dystrophy. Mdx mice fed a 12% NaCl diet from birth up to 20 days of age (mdx-Na mice) had an approximately 50% reduction in serum creatine kinase (CK) activity compared with mdx mice fed a standard diet. Most notably, necrotic fibers in tibialis anterior (TA) muscle of mdx-Na mice were reduced by 99% and were similar in control mice. These mdx mice displayed significantly elevated blood Ca2+ and Na+ levels, while the total calcium content of their TA muscle was reduced to the level of control mice. In addition, mdx-Na mice had elevated zinc and magnesium contents in their TA muscle. These results suggest that elevated serum Na+ leads to Ca2+ extrusion from muscle via the Na+/Ca2+ exchanger causing a decrease in intracellular Ca2+ levels and an increase in blood Ca2+ levels. Extracellular Ca2+ and, in addition, Zn2+ and Mg2+ might also contribute to the stabilization of the cell membrane. Other possibilities explaining the surprisingly efficacious beneficial effect of dietary sodium exist and are discussed.     

Effects of chronic allopurinol therapy on purine metabolism in Duchenne muscular dystrophy

Adenine, adenosine, inosine, hypoxanthine, xanthosine, xanthine, guanine and guanosine blood levels in 11 Duchenne muscular dystrophy patients treated with allopurinol, 10 untreated patients and 8 healthy controls, were determined by HPLC. Serum ADA, PNP and 5'-NT were also determined. Untreated patients showed lower adenine (p less than 0.001) and higher adenosine, xanthine, ADA and PNP levels (p less than 0.01) than controls. Treated patients had lower adenine and higher xanthine levels (p less than 0.001), but higher hypoxanthine, xanthosine and guanine levels (p less than 0.001), than controls, with normal ADA and PNP. The changes observed in ADA and PNP levels suggest an involvement of these enzymes in accelerated degradation of purines in Duchenne dystrophy.     

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.     

Cyclooxygenase-2 expression in skeletal muscle of knockout mice suffering Duchenne muscular dystrophy

The purpose of the present study was to investigate the role of cyclooxygenase-2 (COX-2) expression in fibrotic lesion in mdx mice. A total of six male C57BL/10 mice and six C57BL/10-DMD/mdx were distributed into two groups: control and animals with Duchenne muscular dystrophy (DMD). The medial part of gastrocnemius muscle was evaluated being the specimens stained with hematoxylin and eosin (H&E) and Sirius Red under normal and polarized light to differentiate type I (red and yellow) and III (green) collagen. COX-2 expression was assessed by immunohistochemistry. The results revealed histopathological changes in C57BL/10-DMD/mdx as depicted by regenerating fibers. Sirius Red stain showed a substantial increase in the amount of type I collagen of mdx mice. DMD induced a strong COX-2 immunoexpression in intercellular space. Taken together, our results are consistent with the notion that necrotic and fibrotic lesions are able to increase COX-2 expression in DMD.     

Further observations by electron microscope of striated muscle in progressive Duchenne muscular dystrophy

Gastrocnemius muscle fragments of children affected by clinically diagnosed progressive muscular dystrophy of Duchenne have been studied. At the light microscope, in the semi-thin sections, the more evident changes are represented by a wide diameter range of the fibers and fatty infiltration. Some fibers show numerous nuclei in their central part, a sarcoplasmic degeneration of vacuolar type and an irregular and tortuous course of the myofibrils. Moreover, the ultrastructural findings have shown characteristic changes in myofilaments and Z bands represented by: streaming of the Z bands, collection of the triads and concentric laminated bodies. These observations have pointed out a certain gradualness of the alterations, starting from focal changes of Z band to the complete disarrangement of myofilaments.     

DNA-diagnosis of carriers of the Duchenne muscular dystrophy gene

Duchenne muscular dystrophy carrier detection has been performed by using probes XJ1.1 (intragenic probe) and probe 754 for a girl. The carrier probability was estimated by means of a computer program GenRisk combining pedigree and DNA-probe data and turned out to be 95%.     

Allosteric transition of erythrocyte alkaline phosphatase from Duchenne muscular dystrophy (DMD) patients and Duchenne muscular dystrophy carriers (Homo sapiens)

1. The kinetic properties of the p-nitrophenylphosphatase (EC 3.1.3.1) from erythrocytes was investigated in DMD-patients and DMD-carriers. 2. A different allosteric behaviour in the p-nitrophenylphosphatase from DMD-patients and DMD-carriers compared to controls is supported by the following findings: (a) values of n altered in F- inhibition of (K+)-activated p-nitrophenylphosphatase with Hill coefficients -1.5, -2.2 and -3.1; (b) heterotropic effect of increased concentration of Mg2+ on F- inhibition which is reverted by K+ in DMD-carriers and in control, but not in DMD-patients. 3. Evidence is presented showing that in DMD-patients and in DMD-carriers the interaction membrane-enzyme is different from the corresponding controls.     

The myoblast defect identified in Duchenne muscular dystrophy is not a primary expression of the DMD mutation

Summary We previously proposed the hypothesis that the primary expression of the defect in X-linked Duchenne muscular dystrophy (DMD) occurred in the myoblast, or muscle precursor cell. This was based on the observation that the number of viable myoblasts obtained per gram DMD muscle tissue was greatly reduced and those that grew in culture had decreased proliferative capacity and an aberrant distended flat morphology. Here we test that hypothesis by determining whether the expression of the myoblast defect is X-linked. Muscle cells were obtained from five doubly heterozygous carriers of two X-linked loci, DMD and glucose-6-phosphate dehydrogenase (G6PD), and compared with those from five sex-and age-matched controls heterozygous for G6PD only. A total of 1,355 individual clones were determined to be muscle and evaluated at the single cell level for proliferative capacity, morphology, and G6PD isozyme expression. The results demonstrate that the proportion of defective myoblast clones is significantly increased in DMD carriers. However, since this cellular defect does not consistently segregate with a single G6PD phenotype in the myoblast clones derived from any of the carriers, it is unlikely to be the primary expression of the DMD mutant allele.     

Dystrophin analysis in clonal myoblasts derived from a Duchenne muscular dystrophy carrier.

Clonal myogenic cell cultures were established from a potential heterozygote for a mutant Duchenne muscular dystrophy (DMD) gene who was also heterozygous for isozymes of the X-linked enzyme glucose-6-phosphate dehydrogenase. Previous tissue culture studies of this muscle donor demonstrated equal proliferative capacity of myoblasts that had lyonized either the paternal or maternal X-chromosome, indicating that mutation of the DMD gene does not affect growth of myoblasts. If this muscle donor were a gonadal mosaic, this conclusion would be incorrect. In the present study, only those myogenic colonies expressing the glucose-6-phosphate dehydrogenase-A isozyme were found to express dystrophin, indicating that this woman was indeed a heterozygote for DMD. By documenting dystrophin deficiency in a specific population of myogenic cells from this woman, we verify our previous conclusion regarding the normal proliferative capacity of DMD myoblasts. Somatic cell testing of dystrophin expression may offer an alternative to established genetic carrier tests for those women in whom deletions of the DMD are not detectable, whose pedigree structure does not permit linkage analysis, or in whom standard phenotypic analyses are ambiguous.     

Expression of the gene for large subunit of m-calpain is elevated in skeletal muscle from Duchenne muscular dystrophy patients

Calpain is an intracellular nonlysosomal protease involved in essential regulatory or processing functions of the cell, mediated by physiological concentrations of Ca²⁺. However, in an environment of abnormal intracellular calcium, such as that seen in Duchenne muscular dystrophy (DMD), calpain is suggested to cause degeneration of muscle owing to enhanced activity. To test whether the reported increase in calpain activity in DMD results fromde novo synthesis of the protease, we have assessed the quantitative changes in mRNA specific for m-calpain. mRNA isolated from DMD and control muscle was analysed by dot blot hybridization using a cDNA probe for the large subunit of m-calpain. Compared to control a four-fold increase in specific mRNA was observed in dystrophic muscle. This enhanced expression of the m-calpain gene in dystrophic condition suggests that the reported increase in m-calpain activity results fromde novo synthesis of protease and underlines the important role of m-calpain in DMD.     

Deletions of fetal and adult muscle cDNA in Duchenne and Becker muscular dystrophy patients.

We have isolated a cDNA molecule from a human adult muscle cDNA library which is deleted in several Duchenne muscular dystrophy patients. Patient deletions have been used to map the exons across the Xp21 region of the short arm of the X chromosome. We demonstrate that a very mildly affected 61 year old patient is deleted for at least nine exons of the adult cDNA. We find no evidence for differential exon usage between adult and fetal muscle in this region of the gene. There must therefore be less essential domains of the protein structure which can be removed without complete loss of function. The sequence of 2.0 kb of the adult cDNA shows no homology to any previously described protein listed in the data banks although sequence comparison at the amino acid level suggests that the protein has a structure not dissimilar to rod structures of cytoskeletal proteins such as lamin and myosin. There are single nucleotide differences in the DNA sequence between the adult and fetal cDNAs which result in amino acid changes but none that would be predicted to change the structure of the protein dramatically.