Becker muscular dystrophy is usually caused by intragenic dystrophin gene deletions that result in production of an internally deleted protein. Previous studies have detected what appears to be a unique dystrophin degradation product that appears only in muscle biopsies from patients with Becker muscular dystrophy. This dystrophin fragment is always seen in addition to the "full-size" dystrophin of the expected size for a given gene deletion. It is only found in biopsies from patients with mutations in the deletion-prone region encompassing exons 45-53, but it does not appear to correlate with any observable phenotype at the clinical level. By correlating the size and locations of dystrophin gene deletions with the size of this degradation product, together with use of region-specific dystrophin antisera, we find that proteolytic cleavage may occur at the deletion breakpoints, perhaps due to alterations of the secondary and/or tertiary structures of the protein. This cleavage results in loss of the carboxy-terminal domains that are thought to be important for interactions between dystrophin and other membrane-bound proteins.

A CA dinucleotide repeat polymorphism has been identified for the skeletal muscle alpha-actinin gene ACTN2. The observed heterozygosity is 44% (predicted heterozygosity 50%, PIC 0.47). This polymorphic marker has been localized between D1S74 and D1S103 on the multipoint linkage map of chromosome 1 at a position 44.4 cM from the most distal marker D1S68 at 1 qter.

We report that the glucocorticoid methylprednisolone (Mepd) enhanced myogenesis in normal primary human muscle cultures, but inhibited myogenesis of most Duchenne/Becker muscle cultures. A decline in the magnitude of myogenic stimulation of Mepd correlated with age in a random group of control patients, including some with neurologic diseases other than Duchenne/Becker dystrophy. A case of Duchenne muscular dystrophy from an exceptionally young patient yielded a muscle culture that was myogenically stimulated by Mepd. These results suggest that continuous cycles of degeneration and regeneration of dystrophic muscle in vivo may result in a change of the glucocorticoid response of the muscle progenitor cells. The glucocorticoid effects suggest caution in the long-term clinical use of these agents for muscle disease such as Duchenne muscular dystrophy.

Conserved sequences of dystrophin, beta-spectrin, and alpha-actinin were used to plan a set of degenerate oligonucleotide primers with which we amplified a portion of a human alpha-actinin gene transcript. Using this short clone as a probe, we isolated and characterized full-length cDNA clones for two human alpha-actinin genes (ACTN2 and ACTN3). These genes encode proteins that are structurally similar to known alpha-actinins with approximately 80% amino acid identity to each other and to the previously characterized human nonmuscle gene. ACTN2 is the human homolog of a previously characterized chicken gene while ACTN3 represents a novel gene product. Northern blot analysis demonstrated that ACTN2 is expressed in both skeletal and cardiac muscle, but ACTN3 expression is limited to skeletal muscle. As with other muscle-specific isoforms, the EF-hand domains in ACTN2 and ACTN3 are predicted to be incapable of binding calcium, suggesting that actin binding is not calcium sensitive. ACTN2 was mapped to human chromosome 1q42-q43 and ACTN3 to 11q13-q14 by somatic cell hybrid panels and fluorescent in situ hybridization. These results demonstrate that some of the isoform diversity of alpha-actinins is the result of transcription from different genetic loci.

Duchenne muscular dystrophy patients express little or no dystrophin, while patients with the milder Becker variant produce dystrophin of altered size or quantity. Dystrophin is currently evaluated on Western blots, but quantitation is difficult and the procedure is not available in most clinical laboratories. We describe an enzyme-linked immunosorbent assay (ELISA) for dystrophin that utilizes a carboxyl-terminal capture antibody, and detection antibodies spanning 65% of the molecule. This configuration is selective for dystrophin and reduces the potential for false diagnosis due to loss of antigenic determinants by deletion or the presence of truncated products resulting from frame-shift mutations. The dystrophin ELISA distinguishes Duchenne muscular dystrophy patients from those with unrelated disorders and may have prognostic value for patients with Becker dystrophy. This assay should prove to be an accessible and rapid tool for the diagnosis of Duchenne/Becker muscular dystrophies and for evaluating therapies that attempt to introduce dystrophin or augment its expression.

Nemaline myopathy (NEM) is a neuromuscular disorder characterized by the presence, in skeletal muscle, of nemaline rods composed at least in part of alpha-actinin. A candidate gene and linkage approach was used to localize the gene (NEM1) for an autosomal dominant form (MIM 161800) in one large kindred with 10 living affected family members. Markers on chromosome 19 that were linked to the central core disease gene, a marker at the complement 3 locus, and a marker on chromosome 1 at the alpha-actinin locus exclude these three candidate genes. The family was fully informative for APOA2, which is localized to 1q21-q23. NEM1 was assigned to chromosome 1 by close linkage for APOA2, which is localized to 1q21-q23. NEM1 was assigned to chromosome 1 by close linkage to APOA2, with a lod score of 3.8 at a recombination fraction of 0. Recombinants with NGFB (1p13) and AT3 (1q23-25.1) indicate that NEM1 lies between 1p13 and 1q25.1. In total, 47 loci were investigated on chromosomes 1, 2, 4, 5, 7-11, 14, 16, 17, and 19, with no indications of significant linkage other than to markers on chromosome 1.

Abnormalities of dystrophin, a cytoskeletal protein of muscle and nerve, are generally considered specific for Duchenne and Becker muscular dystrophy. However, several patients have recently been identified with dystrophin deficiency who, before dystrophin testing, were considered to have Fukuyama congenital muscular dystrophy (FCMD) on the basis of clinical findings. Epidemiologic data suggest that only 1/3500 males with autosomal recessive FCMD should have abnormal dystrophin. To explain the observation of 3/23 FCMD males with abnormal dystrophin, we propose that dystrophin and the FCMD gene product interact and that the earlier onset and greater severity of these patients' phenotype (relative to Duchenne muscular dystrophy) are due to their being heterozygous for the FCMD mutation in addition to being hemizygous for Duchenne muscular dystrophy, a genotype that is predicted to occur in 1/175,000 Japanese males. This model may help explain the genetic basis for some of the clinical and pathological variability seen among patients with FCMD, and it has potential implications for understanding the inheritance of other autosomal recessive disorders in general. For example, sex ratios for rare autosomal recessive disorders caused by mutations in proteins that interact with X chromosome-linked gene products may display predictable deviation from 1:1.

We present two cases of autosomal dominant limb girdle muscular dystrophy in a father and son. Both presented in childhood with a classical Becker muscular dystrophy phenotype. The father had initially been informed that he would not have affected children. After the diagnosis of muscular dystrophy in the son, immunoblot analysis was performed on muscle and revealed normal dystrophin. The polymerase chain reaction did not show any deletions in the dystrophin gene, and the father's dystrophin gene was not passed to his son. These cases demonstrate that autosomal dominant muscular dystrophy may present in childhood, and that dystrophin and molecular genetic analyses should be performed when considering the diagnosis of childhood muscular dystrophy, even in the presence of a classical phenotype.

Becker muscular dystrophy (BMD) often results from in-frame mutations of the dystrophin gene that allow production of an altered but partially functional protein. To address potential structure-function relationships for the various domains of dystrophin, we examined both the dystrophin gene and protein in 68 patients with abnormal dystrophin. Eighty-six percent of BMD patients with dystrophin of altered size have deletions or duplications, and the observed sizes of dystrophin fit well with predictions based on DNA data. Deletions within the amino-terminal domain I tended to result in low levels of dystrophin and a more severe phenotype. The phenotypes of patients with deletions or duplications in the central rod domain were more variable. This region can be divided into three portions based on differences in clinical presentations of patients. Deletions around exons 4553 were most common and generally caused typical BMD; however, phenotypic variability among patients with similar mutations suggests that epigenetic and/or environmental factors play an important role in determining the clinical progression. In contrast, deletions or duplications in the proximal portion of this domain tended to cause severe cramps and myalgia. Finally, loss of the middle of this region probably causes a very mild phenotype, as only one such patient was found and his only symptom was elevated serum creatine phosphokinase levels.

Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is expressed in brain as well as muscle. The role of dystrophin in the brain is not clear, though one-third of Duchenne muscular dystrophy patients exhibit some degree of mental retardation. We have isolated the genomic region encoding the alternative 5' terminus of dystrophin used in the brain. Primer extension and polymerase chain reaction assays on RNA demonstrate that this region contains an alternative promoter for dystrophin used in the brain. Physical mapping of this region indicates that this brain promoter is located greater than 90 kilobases 5' to the promoter used in muscle and 400 kilobases from exon 2 to which it is spliced. The large physical distance between the promoters, taken together with their known tissue selectivities, suggests that in certain patients a deletion of either dystrophin promoter might give rise to reduced dystrophin expression selective to brain or muscle. We have identified one such individual with specific deletion of the dystrophin muscle promoter, giving rise to Becker muscular dystrophy, and we predict that specific loss of the brain promoter may be one cause of X chromosome-linked mental retardation.